Lipoparticles comprising proteins, methods of making, and using the same

ABSTRACT

The present invention relates to lipoparticles. The invention also relates to producing lipoparticles. The invention further relates to lipoparticles comprising a viral structural protein. The invention further relates to a lipoparticle comprising a membrane protein, and the lipoparticle can be attached to a sensor surface. The invention further relates to methods of producing and using the lipoparticle to, inter alia, assess protein binding interactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/491,477, filed Jul. 30, 2003, U.S. Provisional Application Ser.No. 60/491,633, filed Jul. 30, 2003, U.S. Provisional Application Ser.No. 60/498,755, filed Aug. 29, 2003, U.S. Provisional Application Ser.No. 60/502,478, filed Sep. 12, 2003, U.S. Provisional Application Ser.No. 60/509,677, filed Oct. 7, 2003, U.S. Provisional Application Ser.No. 60/509,608, filed Oct. 7, 2003, and U.S. Provisional ApplicationSer. No. 60/509,575, filed Oct. 7, 2003, each of which is hereinincorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support (NIH Grants No.GM64924, GM68322, and RR16832) and the U.S. Government may thereforehave certain rights in the invention.

BACKGROUND OF THE INVENTION

Ligand interactions with membrane proteins are responsible for amultitude of cell adhesion, signaling, and regulatory events. Thisdiversity of function makes membrane proteins important drug targets.G-protein coupled receptors (GPCRs) are one family of membrane proteinsthat comprise nearly half of existing drug targets (Drews (1996), Nat.Biotechnol., 11:1516-1518, Stadel, et al. (1997), Trends Pharmacol.Sci., 18:430-437, Wise, et al. (2002), Drug Discovery Today, 7:235-246).Another 5% of drug targets are comprised of membrane-embedded ionchannels, and many of the remaining targets are also membrane proteins.These topologically complex membrane proteins span the lipid bilayer ofthe cell multiple times and usually serve as receptors to mediatecommunication between a cell's function and its exterior environment.Drugs targeting membrane proteins include antipsychotics,antihistamines, beta-blockers, anti-migraine drugs, anti-ulcer drugs,and analgesics.

Despite their importance, proteins that span the membrane multiple timespresent a unique set of challenges for ligand binding and drug discoverystudies because they require a lipid environment to maintain nativestructure. For example, GPCRs weave in and out of the cell membraneseven times, so cannot be extracted from the lipid bilayer withoutdisrupting their structure. While detergent conditions can occasionallybe found that allow native structure to be maintained in solution, thisis an empirical and very time-consuming process, and even then stabilityis only transient.

As a result, interaction studies involving membrane proteins typicallyuse whole cells or vesicles derived from cell membranes, where theprotein of interest is but a minor component, making both adequatesensitivity and specificity more difficult to achieve. Living cells arecumbersome to grow, must be maintained in a high protein (serum)environment, and present a moving target as receptors are internalized,altered by intracellular events, and recycled. Cells also have severelimitations in their application to biosensors and other microfluidicdevices, most notably in their size, sensitivity to environmentalconditions, and heterogeneous cell surface. Membrane vesicles, preparedby mechanically disrupting cells, are a common source of membraneprotein for many drug screens currently conducted. However, membranevesicles are heterogeneous, impure, and not particularly stable. Thereceptors within them may be misoriented, a minor component of totalprotein, and derived from intracellular organelles.

Thus, there remains a need for the development of a broad methodologythat permits the rapid purification of a wide spectrum of membrane andcellular proteins. The present invention satisfies this need. Further,there is a need for assays that permit the study of membrane proteininteractions, and the present invention also satisfies this need.Further, there is a need for vesicles that facilitate the elicitation ofan immune response against membrane proteins to be mounted, particularlyfor the generation of monoclonal antibodies, humoral response, cellularresponse, and vaccines, and the present invention also satisfies thisneed. The present invention fulfills these needs as well others.

Human pathogens enter their host cells, and eventually kill or weakenthem, using cellular receptors. In nearly all cases, these cellularreceptors are membrane proteins on the cell surface. Identifying thesemembrane proteins and linking infectious agents to their receptorsoffers direct insight into disease pathogenesis. For example, HIV uses afusion co-receptor, typically either the chemokine receptors CXCR4 orCCR5 (Doranz (2000), Emerging Therapeutic Targets, 4:423-437). Strainsof HIV that use the co-receptor CXCR4 are correlated with increaseddisease pathogenicity, while strains that use the co-receptor CCR5 areresponsible for transmission of the virus. Preventing receptor bindingis a common strategy for treating or preventing pathogen infection.

Cellular receptors are not only involved in pathogen binding, fusion,and entry, but are also involved with other pathogenic processes. Forexample, many pathogens encode toxins that facilitate virulence of thepathogen. Examples include anthrax toxin (Bradley, et al. (2003),Biochem Pharmacol, 65:309-314), HHV8-encoded chemokines (vMIP-2) (Holst,et al. (2003), Contrib Microbiol, 10:232-252), cytomegalovirus chemokineUL146 (Penfold, et al. (1999), Proc. Natl. Acad. Sci. USA,96:9839-9844), and pox virus-encoded chemokine inhibitors (vCCI, M-T1,M-T7) (Lalani, et al. (1997), J. Leukoc. Biol., 62:570-576, Smith, etal. (1997), Virology, 236:316-327). In many cases, identifying andinhibiting these toxins and their receptors can explain the lethaleffects of the pathogen and neutralize their actions.

The techniques currently used to identify pathogen receptor interactionsare generally slow and laborious. In many cases, such techniques requiretransfection of individual receptors, the growth of cells, and the useof the pathogen in its infectious form. In other cases, proteininteractions are detected using immunoprecipitation, Western blotting,or radiolabeled proteins. For example, the HIV co-receptors CCR5 andCXCR4 were identified over ten years after HIV and its primary receptor,CD4, were discovered.

Optical biosensors are a class of instruments that can detect affinitiesof intramolecular interaction from picomolar to micromolarconcentrations, in real-time, and without labels. Biosensors can alsoyield pharmacological information (kinetic and equilibrium bindingconstants) that other assays cannot (see (Canziani, et al. (1999),Methods, 19:253-269, Day, et al. (2002), Protein Science, 11:1017-1025)for review). Biosensors have been integrated into both drug discoveryand diagnostics.

The most widely used optical biosensor, the BIACORE™, is based onsurface plasmon resonance (SPR), which measures changes in refractiveindex at the sensor surface. The BIACORE platform consists of a flowcell with three inert walls (sides and floor) and a gold ceiling that ischemically modified to attach biomolecules. During usage, binding ofprotein in solution to tethered protein on the biosensor surface ismonitored by changes in refractive index at the chip surface. A numberof new biosensor devices are emerging that operate on similarprinciples.

Biosensors can also operate by measuring changes in spectroscopicmeasurements, such as reflectance, absorbance, transmission, orresonance. For example, microcantilever-based biosensors operate bydetecting mechanical deflections of light reflecting frommicrocantilevers. The microcantilevers can be conjugated with anantibody, a protein, a ligand, a small molecule, a peptide, or alipoparticle. The binding partner that deflects the cantilever can alsobe an antibody, a protein, a ligand, a small molecule, a peptide, or alipoparticle.

Other biosensors are based on surface plasmon resonance and operateusing an array format (on the world wide web at htsbiosystems.com).Other biosensors are based on colorimetric diffraction grating andoperate using an array format (Cunningham, et al. (2002), Sensors andActuators, B81:316-328, Cunningham, et al. (2002), Sensors andActuators, B85:219-226, Lin, et al. (2002), Biosens Bioelectron,17:827-834). Other biosensors are based on acoustic resonators (Cooper,et al. (2001), Nat Biotechnol, 19:833-7).

Such biosensors, however, have not been widely suitable for membraneproteins because: 1) biosensor detection signals are a function ofdistance from the surface and structures larger than 200 nm yield poorsignals (e.g. cells, membrane vesicles), 2) the purity of moleculestethered to the biosensor surface is directly proportional to thesignal-to-noise ratio, 3) the use of microfluidic channels limits thesize of flow components, 4) the nature of biosensor detection restrictsnearly all such devices to soluble molecules, and 5) removal of membraneproteins from their native lipid environment destroys their structure.

Only a handful of proof-of-concept studies have detected binding tomembrane proteins using optical biosensors, but not using lipoparticles(Bieri, et al. (1999), Nat. Biotechnol., 17:1105-1108, Cooper, et al.(2000), Analytical Biochemistry, 277:196-205, Cooper, et al. (1998),Biochim Biophys Acta, 1373: Heyes, et al. (1998), Biochemistry,37:507-522, Karlsson, et al. (2002), Analytical Biochemistry,300:132-138, Salamon, et al. (2000), Biophysical Journal, 79:2463-2474,Salamon, et al. (1994), Biochemistry, 33:13706-13711, Salamon, et al.(1996), Biophysical Journal, 71:283-294). All of these studies useddetergent-solubilized membrane proteins, and most focused on oneprototype protein where solubilization conditions have been well studied(rhodopsin). Detergent-solubilized membrane proteins have been used toinvestigate membrane protein interactions, although this approach hassignificant drawbacks. As discussed previously, G-protein coupledreceptors (GPCRs) weave in and out of the cell membrane seven times, andthus cannot be extracted from the lipid bilayer without disrupting theirstructure.

Doms et al. (U.S. Patent Publication 2002/0183247 A1) discusses usingbiosensors to detect interactions between membrane proteins onlipoparticles and their binding partners, however improvements are stillneeded.

Thus, there remains a need for improved methods for using membraneproteins with optical biosensors. One aspect of the present invention isto use arrays of membrane proteins in conjunction with opticalbiosensors to recreate the cell surface in vitro. This will result in aproduct that will consist of a biosensor chip containing the thousandsof membrane proteins encoded by the human genome. There is also a needfor improved methods and techniques to identify receptors for viralentry. There is also a need for improved methods of identifyingreceptors for ligands or drugs. There is also a need for improvedmethods of identifying unknown pathogens or substances in a sample. Thepresent invention satisfies these needs and others.

Biological probes comprise one or both of two basic functional features:a targeting component and a reporting component. The targeting componentinteracts with the structure or molecule of interest and defines theability of the probe to discriminate target structures or events. Thereporting component signals target interaction and defines detectionparameters such as sensitivity. Most biological probes in current use(e.g. antibodies, fluorescent proteins, ion-sensitive dyes) are singlemolecule structures, and thus usually possess a single reporting domainand either no or one targeting domain.

Molecules designed to detect the presence of specific biological targetsor report the occurrence of biological events are known broadly asmolecular probes. Traditional imaging probes comprise a recognitioncomponent (in this context defined as a “targeting” domain) which bindsto a target molecule, and a signaling component (in this context definedas a “reporter”) which illuminates it (reviewed in (Massoud, et al.(2003), Genes Dev, 17:545-80, Molecular Probes Handbook (2003), Zhang,et al. (2002), Nat Rev Mol Cell Biol, 3:906-18)). The most widely usedreporters emit an electromagnetic signal in the visual spectrum(bioluminescence or fluorescence), but radioactive and magnetic signalsare also of medical importance for imaging techniques such asautoradiography, positron emission tomography (PET), and magneticresonance imaging (MRI). Examples of visual reporters includefluorescent proteins, luminescent substrates, quantum dots, andfluorescent dyes. Biological probes incorporating these types ofreporters are used to physically map cell structures and tissuearchitecture, as well as to monitor (and correlate) biologicalfunctions. The cellular structures and events that are emerging asimportant targets of molecular probes include subcellular componentsinvolved in gene transcription, cell growth and proliferation, cellmigration and second messenger pathways.

With the increasing need to monitor smaller targets, and to dissectcomplex cellular functions at greater resolution, has arisen arequirement for the development of more sophisticated probes withimproved localization and reporting characteristics. Cells are able tointerpret the meaning of individual signaling events, for example,because each is part of a larger cellular response which includessequential second messenger activity and spatial localization ofsignaling components. In contrast, most of the probes used in researchand diagnostics to interpret such cell events rely on simple end-pointmeasurements of a single event, target, or phenomenon (e.g. fluctuationsin cytosolic calcium), and often fail to resolve the target to asub-cellular location. The two most important characteristics ofemerging biomedical imaging strategies are the ability to localize andalign structural and functional information at tissue, cellular andsub-cellular levels, and the ability to exploit ‘multimodal’ detectionsystems (more than one reporter being simultaneously detected orcorrelated). Improvements in these properties can allow superior spatiallocalization of abnormalities in vivo, as well as structure-functioncorrelation on the subcellular level (Massoud, et al. (2003), Genes Dev,17:545-80). These ‘new generation’ probes are playing an increasinglyimportant role in defining molecular events in the fields of cancerbiology, cell biology, and gene therapy, for example in the detection oftumor markers and in tracking the delivery and function of gene therapyvectors (Jendelova, et al. (2004), J Neurosci Res, 76:232-43, Ray, etal. (2003), Cancer Res, 63:1160-5, Townsend, et al. (2001), Eur Radiol,11:1968-74, Wu, et al. (2003), Nat Biotechnol, 21:41-6). Most existingsingle-molecule probes possess inherent limitations in these propertiesdue to difficulties in integrating complex targeting and reportingsystems. Although multiple probes can be introduced to simultaneouslydetect several events, most reporters cannot be targeted to desiredcellular or subcellular structures to more accurately differentiatesignaling events. Those probes that can be localized (e.g. fluorescentantibodies) usually contain only a small number (1-4) of reporterfluorophores per molecule, limiting signal amplification and detection.

A major obstacle in constructing improved imaging probes is not simplythe development of new reporter molecules, but the development of asuitably sophisticated format in which complex and multiple reporterscan be linked, and, importantly, controlled for target localization.Probes that can be spatially localized at the nanometer scale, that canamplify infrequent signals, and that can compartmentalize multiplereporters simultaneously, could have a major impact on developingsubcellular and nano-scale applications in biomedical research anddiagnostics.

A variety of foreign soluble proteins can also be incorporated intoretroviruses. The incorporation of green fluorescent protein (GFP) intoretroviruses has been used in a number of studies to understand aspectsof the retroviral lifecycle such as budding, assembly, and infection(Andrawiss, et al. (2003), J Virol, 77:11651-60, Dalton, et al. (2001),Virology, 279:414-421, McDonald, et al. (2002), J. Cell Biol., 159:McDonald, et al. (2003), Science, 300:1295-7). In addition, labeling ofviruses with fluorescent reporters has been used on several occasions tounderstand the early stages of virus fusion, endocytosis, and nuclearmigration (Bartlett, et al. (1998), Nat Med, 4:635-7, Leopold, et al.(2000), Hum Gene Ther, 11:151-65, McDonald, et al. (2002), J. CellBiol., 159: McDonald, et al. (2003), Science, 300:1295-7). An additionalstudy demonstrated proof-of-concept incorporation of a different foreignprotein fused to Gag (cytochrome c from yeast) (Weldon, et al. (1990), JVirol, 64:4169-79, Wills (1989), Nature, 340:323-4; U.S. Pat. No.5,175,099). Probes attached to viruses have been limited to antibodiesor fluorescent proteins that typically have limited life-spans and havenever reported anything more than the location of the virus. Similarwork has also been performed to study the phagocytosis of fluorescentyeast and bacteria (a product currently sold by Molecular Probes as‘BioParticles’) (Giaimis, et al. (1994), Cytometry, 17:173-8, Haugland(2003), Oben, et al. (1988), J Immunol Methods, 112:99-103, Perticarari,et al. (1994), J Immunol Methods, 170:117-24, Ragsdale, et al. (1989), JImmunol Methods, 123:259-67), but virus-based bioparticles have neverbeen developed. The incorporation of proteins of desired specificity andfunction into retroviruses has never been pursued for probe purposes.

There has been a desire and long-felt need for vehicles that canencapsulate and target multimodal probes to be used in the imaging field(Massoud, et al. (2003), Genes Dev, 17:545-80). The use of liposomes andrelated lipid structures is one approach that others have pursued. Forexample, synthetic beads conjugated with lipids have been used to createfluorescent sensors for pH, chloride, calcium, and oxygen that have beenused to probe intracellular compartments and phagocytic pathways (acritical component of the immune response against infections) (Ji, etal. (2000), Anal Chem, 72:3497-503, Ji, et al. (2001), Anal Chem,73:3521-7, Ma, et al. (2004), Anal Chem, 76:569-75, McNamara, et al.(2001), Anal Chem, 73:3240-6, Nguyen, et al. (2002), Anal Bioanal Chem,374:69-74). However, the implementation of such probes has generallybeen limited to materials (usually synthetic dyes) that can beencapsulated within a lipid bilayer. Functional enzymes and membraneproteins have been much more difficult to capture within such structures(Walde, et al. (2001), Biomol Eng, 18:143-77). Moreover, synthetic lipidvesicles are difficult to localize and often lack the stabilitynecessary for wide-spread application.

The specificity of membrane proteins can be controlled, in part, byengineering membrane proteins to bind antigen-specific antibodies. Theantibody-binding (Z) domain of Staphylococcal Protein A (ProA) binds theconstant, Fc, domain of IgG. Membrane-anchored antibodies andantibody-containing structures (ZZ-TM fusion proteins) have beenincorporated into cells and viruses, primarily for use in targeting ofgene therapy vectors (Bergman, et al. (2003), Virology, 316:337-47,Masood, et al. (2001), Int J Mol Med, 8:335-43, Morizono, et al. (2001),J Virol, 75:8016-20, Nakamura, et al. (2004), Nat Biotechnol, 22:331-6,Ohno, et al. (1997), Biochem Mol Med, 62:123-7, Ohno, et al. (1997), NatBiotechnol, 15:763-7, Sawai, et al. (1998), Mol Genet Metab, 64:44-51,Snitkovsky, et al. (2000), J Virol, 74:9540-5). In all of the publishedcases, ZZ-TM fusion proteins bound antibody in the appropriateorientation for targeting of retroviruses to cells expressingcomplementary antigen.

The use of fluorescently-labeled beads as a solid substrate for bindingreactions has recently become a popular replacement for traditional96-well plates in such techniques as immunoassays and competitivebinding assays. By flowing beads and their associated protein reactantsthrough a kinetic fluidics system, and by incorporating into the beads anumber of different dyes that can be detected after excitation atdifferent wavelengths of light, sample throughput has been increased byallowing the simultaneous detection of multiple analytes (multiplexanalysis).

Thus, there is a need for improvements in the performance of probes inthe areas described above to allow superior spatial localization ofabnormalities in vivo, as well as structure-function correlation on thesubcellular level, and already have applications in the fields of cancerbiology, cell biology, and gene therapy, for example in the detection oftumor markers and in tracking the delivery and function of gene therapyvectors. There is also a need for probes that can be spatiallylocalized, that can amplify infrequent signals, and that cancompartmentalize multiple reporters simultaneously. There is also a needto generate probes that can generate multimodal signals. The presentinvention satisfies the needs as well as others.

Ion channels are membrane-bound proteins which control the flow of ionsacross biological membranes, either through passive or active transportmechanisms. In the context of cell electrophysiology, ion channels arethe primary molecular mechanism by which cells maintain a membranepotential. Membrane potential is generated and maintained byconcentration gradients of charged ions such as sodium, potassium,chloride, hydrogen, and calcium, across the otherwise impermeable cellmembrane. The membrane potential of a cell can change in the course ofsignaling, development, differentiation of function, and pathology.

Electrical potential differences are present across the plasma membraneof most living prokaryotic and eukaryotic cells, and also between thecytosol and the interior of organelles such as chloroplasts andmitochondria. As a consequence of ion concentration gradients that aremaintained by active transport processes, the electrical membranepotential of some resting cells is approximately −70 mV, with the cellinterior electrically negative with respect to the exterior. Themembrane potential is reduced to zero when the plasma membrane isruptured by chemical or physical agents. When a membrane is permeable toonly a single ion species (the simplest theoretical model), the membranepotential is given by the Nernst equation:V=−(RT/zF)*ln([I]_(in)/[I]_(out)) where R is the gas constant, T is theabsolute temperature, z is the ion valency, F is Faraday's constant, Iis the cation concentration. The value of RT/F is 25.7 mV at 25° C.

A change in membrane potential in the positive direction is called adepolarization. Conversely, a change in membrane potential in thenegative direction is called a hyperpolarization. Depolarization of thecell membrane during the action potential of a nerve or muscle celltypically results in the cell interior transiently becoming electricallypositive with respect to the exterior, as Na-channels open, Na+ rushesin, and membrane potential approaches the V_(ion) of Na+, +60 mV.Voltage-gated K-channels will open when there is sufficientdepolarization, allowing K+ to rush out and bringing the membranepotential back to its resting value, approximately the V_(ion) of K+.

Membrane potential is often measured by electrophysiological methods inwhich glass microelectrodes are inserted into or onto cells (e.g.voltage clamp, patch clamp) to directly measure the difference inelectrical potential across the cell membrane. Many biologicalstructures, however, are not readily amenable to microelectrodemeasurement, such as sub-cellular organelles and neuronal processes.Moreover, microelectrode techniques are difficult to automate for drugdiscovery applications.

In these cases, fluorescent dyes and probes that measure electricalmembrane potential represent an alternative. Fluorescent dyes that canmeasure membrane potential were first employed in the 1970s, and haveevolved in capability, speed of response, and sensitivity for the pastthirty years (Cohen, et al. (1978), Rev Physiol Biochem Pharmacol,83:35-88, Molecular Probes Handbook (2002), Plasek, et al. (1996), J ofPhotochemistry and Photobiology, 33:101-124, Smith (1990), BiochimBiophys Acta, 1016:1-28). Numerous studies have shown that optically andelectrically recorded action potentials are identical in shape, andfluorescent dyes are now a well-accepted methodology for measuringelectrical potential across a membrane. Since a single ion channel canfacilitate the flux of a million ions per second, membrane potentialchange can be detected with high sensitivity even in cases of limitedquantities of ion channel.

Many biological compartments, despite their importance in biology, arenot readily amenable to ion or voltage measurements using conventionaltechnologies. For example, some intracellular organelles are too smallto seal with a microelectrode and cannot be readily labeled with afluorescent dye or probe independent of the rest of the cell. Similarly,synaptic junctions are dynamic structures with budding vesicles,interstitial gaps, and secreted neurotransmitters and ions that cannotbe easily isolated from the structure as a whole.

Thus, there remains a need for the development of an improvedmethodology that permits the study of ion channels and/or transporterproteins. The present invention satisfies this need. Further, there is aneed for assays that permit the study of ion channel inhibitors and/oractivators, and the present invention also satisfies this need as wellothers.

Viral vaccines are made in several ways, but all are significantlydifferent than the lipoparticle. At least half a dozen differentsuccessful virus vaccine systems (summarized in Table 1) give supportfor using small particles to induce immune responses. Lipoparticles areunique because they are able to incorporate membrane proteins which arenot part of the viral genome.

TABLE 1 Killed virus vaccines (Field's Virology). Enveloped indicateswhether virus is surrounded by a lipid bilayer. Virus Vaccine EnvelopedType of Vaccine Hepatitis A − Whole inactivated virus Hepatitis B +Recombinant virus-like particles Influenza A and B + Disrupted virusJapanese encephalitis virus + Whole inactivated virus Poliovirus − Wholeinactivated virus Rabies + Whole inactivated virus

Parvovirus-like particles (VLPs) and have been used to induce cytotoxicT lymphocyte (CTL) responses. This technology involves linking anantigen to the viral particle, which is significantly different than thelipoparticle which incorporates a membrane protein of interest into thelipid membrane of the cell. The Parvovirus VLP technology does notinvolve a lipid bilayer and therefore cannot allow a membrane protein tomaintain its structure. Martinez et al (2003) used a recombinant VLPcarrying a CD8(+) T cell determinant to successfully induce an immuneresponse in mouse neonates (Martinez et al (2003) Virology. 2003; Jan.20; 305(2):428-35). Wakabayashi et al (2002) fused the HPV16 E7 antigento the major and minor capsid proteins (L1 and L2) of chimeric humanpapillomavirus (HPV) virus-like particles (cVLPs) (Wakabayashi et alIntervirology. 2002; 45(4-6):300-7). Mice vaccinated with this cVLPsuccessfully generated a specific CTL response.

Antibodies are also useful in determining structure and function of apolypeptide, and can also be used as therapeutics. Polyclonal antibodiesare a mixture of many antibodies recognizing different epitopes of anantigen. It is possible to isolate one of these antibodies and amplifyit by fusing the antibody cell to an immortal tumor cell, forming ahybridoma. Amplification of the hybridoma results in a clonal populationof cells that secrete antibodies which are identical; these antibodiesrecognize the same epitope on the same antigen, and are known asmonoclonal antibodies (Mabs). Because of their specificity to a singleepitope, Mabs that recognize non-linear epitopes (epitopes formed by aconformational structure of sequentially separated amino acids) aresensitive probes of subtle conformational changes brought about byeither mutational alterations or variations in physical/chemicalconditions.

Historically, it has been difficult to generate good Mabs to manyintegral membrane proteins. Traditional methods of immunization usingpurified proteins or peptides have limited application to membrane-boundreceptors in which removal of the protein from a lipid environmentresults in complete loss of conformation. While antibodies can beelicited against peptides derived from extracellular sequences oftopologically complex receptors, such antibodies often react with thenative receptor inefficiently. The use of whole cells has been used withsuccess for the development of some antibodies to complex receptors(Lee, et al. (1999), J. Biol. Chem., 274:9617-9626), but has a number oflimitations. For example, many receptors, especially when over expressedwithin a cell, can reduce cell health, cause cell death, be cycled awayfrom the cell surface, aggregate, denature, or have difficulty beingtranslated, leading to poor expression. In addition, the protein ofinterest is typically a minor component on the cell surface, and thenumbers of Mabs elicited by this approach is typically small.

The chemokine receptors CCR5 and CXCR4 serve as examples of integralmembrane proteins which are difficult to generate Mabs against. CCR5 andCXCR4 are GPCRs that in addition to their normal functions in the immunesystem are also used by HIV to infect cells. Due to their importance forvirus infection, considerable effort has been spent on developingimmunological reagents to these and related receptors. Some of the firstCCR5 and CXCR4 receptor antibodies were obtained via immunization withpeptides (Doranz, et al. (1997), J. Virol., 71:6305-6314). However,second-generation antibodies obtained following immunization of micewith cells over-expressing the receptor of interest supplanted thesefirst generation antibodies (Lee, et al. (1999), J. Biol. Chem.,274:9617-9626). While such an approach is labor intensive and only avery small fraction of hybridomas target the desired receptor, it is nowclear that presentation of GPCRs in their native conformation isessential for the generation of effective Mabs. The solved structure ofbovine rhodopsin, a 7 transmembrane (7TM) receptor, as well asstructure-function studies on a variety of 7TM receptors including CCR5and CXCR4, indicate that the extracellular domains of these proteins areconformationally complex. In addition, disulfide bonds link the aminoterminal domain of these receptors with the third extracellular loop,and the first with the second extracellular loops. Thus, it is notsurprising that the epitopes recognized by many Mabs are composed ofresidues from multiple extracellular domains of the receptor. Therefore,the native conformation of the receptor as it resides in the membranecan be of critical importance not just for its function, but also forthe elicitation of specific, high affinity Mabs.

The antigenic structures of topologically complex proteins such asGPCRs, amino acid transporters, and ion channels are criticallyinfluenced by the lipid environment; a large fraction of the protein'smass, sometimes even a majority, is embedded in the lipid bilayer.Attempts to reconstitute such receptors in artificial membranes haveproven difficult, though not impossible. In our experience, the mostuseful Mabs to 7TM proteins have thus far come from using cells forimmunization that express the receptors in their native conformation.While this approach has proven successful for the generation of Mabs tothe chemokine receptors CXCR4, APJ, CCR2, CCR3, and CCR5, it has provendifficult for the generation of Mabs to the chemokine receptors XCR1(the Lymphotactin receptor), CCR4, CCR7, CCR8, and CX3CR1 (theFractalkine receptor). In addition, most attempts have failed to producehigh affinity Mabs to topologically complex receptors such as glucosetransporters (e.g. Glut4), ion channels (e.g. Kv1.3), and amino acidtransporters (e.g. MCAT1).

Thus, there is a need for compositions that facilitate the elicitationof an immune response against native membrane protein structures,particularly for the generation of monoclonal antibodies, of humoralresponse, of cellular response, and for vaccines, and the presentinvention also satisfies this need. The present invention fulfills theseneeds as well others.

Protein transfection is defined as the internalization of an exogenousprotein into a target cell's cytoplasm or nucleus. While similar to DNAtransfection in that both processes ultimately introduce exogenousprotein into a cell, protein transfection differs by bypassing thetranscription and translation machinery needed for protein productionfrom DNA. Furthermore, by introducing a foreign protein directly intothe target cell, protein transfection introduces the protein faster thanDNA transfection and bypasses potential complications of DNAtransfection and protein synthesis, such as undesired or incorrect RNAsplicing variations, protein folding, and post-translationalmodifications.

Current applications for protein transfection include studying proteinsthat are inherently toxic to cells, that are involved in signalingcascades within cells, and that are downstream cascade regulators. Forexample, antibodies transfected into the cytoplasm can be used toinhibit or modify downstream signaling cascade effectors (Marrero 1995;Rui 2002). Additionally, fluorescent ligands and substrates can betransfected into a target cell's cytoplasm or nucleus to study asignaling pathway of interest (Nolkrantz 2002).

Previous protein transfection techniques include microinjection,myristylation of amino termini, protein encapsulation by various lipidformulations, such as those comprising cationic lipids, and the use ofpeptides that bind to the protein that is being transfected (e.g.Protein Transduction Domains (PTDs). Current commercial kits includePro-Ject (Pierce Biotechnology, Inc.) and Profect (Targeting Systems)both using lipid-based technologies, and Chariot™, protein deliveryreagent, (Active Motif) using PTD technology. Other methods of proteintransfection include the HIV Tat protein. However, current methods stilldo not resolve the problems of transfecting membrane proteins into acell. Most, if not all, membrane proteins require proper folding andstructure to be active. The current methods of transfecting membraneproteins are inadequate and can be improved.

Thus, there is a need for new and improved protein transfection methodsand compositions to introduce proteins into cells. The present inventionfulfills this need as well as others.

Viruses are of great significance to the field of medicine, but thereare few techniques for quantifying and characterizing viruses due totheir exceptionally small size. Most viruses are between 30 nm and 1 μm,too small for direct visualization under a light microscope or with theuse of most cellular detection methods. Quantification of viruses can beespecially difficult, with most researchers relying on infectious assaysfor quantification, an assay that typically takes several days, requireslive virus, and detects only infectious virions. Many infectious virusesare produced along with 10 to 100-fold more non-infectious viruses, andthese non-infectious viruses are rarely quantified (Knipe, et al.(2001)). Visualization of fluorescent viruses has been used in somecases to overcome these obstacles (Leopold, et al. (2000), Hum GeneTher, 11:151-65, McDonald, et al. (2002), J. Cell Biol., 159: McDonald,et al. (2003), Science, 300:1295-7, Seisenberger, et al. (2001),Science, 294:1929-32).

In addition, the proteins that reside on the surface of lipid-envelopedviruses are also difficult to detect and quantify. Previous research hasdisclosed methods of detecting some membrane proteins on the surface ofintact HIV and SIV virions (Bastiani, et al. (1997), J. Virol.,71:3444-3450, Capobianchi, et al. (1994), J Infect Dis, 169:886-9,Nyambi, et al. (2001), J Immunol Methods, 253:253-62, Orentas, et al.(1993), AIDS Res Human Retroviruses, 9:1157-1165). In addition, researchhas demonstrated the detection of membrane proteins on a virus-likeparticle using confocal microscopy (Zemanova, et al. (2004),Biochemistry). However, many membrane proteins can be relatively sparse,difficult to detect, and even more difficult to quantify on an absolutebasis (Coorssen, et al. (2002), Anal Biochem, 307:54-62). Yet suchmembrane proteins, such as the native viral envelope protein on HIV,gp160, can be central to the infectious life cycle of the virus and thepathogenesis of the disease it causes.

Furthermore, membrane proteins compose a complex structure that is oftendependent on the lipid bilayer in which they reside. Methods to measurethe structural state of the membrane protein within an enveloped virusare needed. When the protein of interest is Envelope, infection assayshave been used, but in cases where the membrane protein does not mediateviral entry other methods are needed.

Currently, the most commonly employed assays for protein interactionsinclude radioimmunoassays, competitive protein binding assays, andenzyme-linked immunoassays. All of these, however, suffer from a numberof drawbacks: they are expensive, labor-intensive, time-consuming, andhave not been used to study membrane proteins without the use of wholecells or membrane vesicles derived from cells. One of the majorlimitations to many in vitro analyses of protein binding is therequirement for the protein(s) of interest to be structurally intact andin solution in order for appropriate interaction with receptor to occur.Many proteins of interest, including the receptors of many viralenvelope proteins, are integrated into cell membranes. These proteinsare notoriously difficult to purify and solubilize in theirstructurally-intact form, limiting the ability to work with them. Assuch, laboratory techniques involving such proteins are eithernon-existent or involve laborious preparatory steps.

Thus, there remains a need for new and/or improved methods for detectingand quantifying viruses, viral particles, virus like particles andlipoparticles. There is also a need for new and/or improved methods andtechniques to detect and quantify the membrane proteins that reside inviruses, viral particles, and lipoparticles. There is also a need forimproved methods of identifying proteins, antibodies, ligands, or drugsthat bind to these membrane proteins. The present invention satisfiesthese needs and others.

Sensors capable of detecting infectious agents and/or of detecting aserological reaction in people exposed to dangerous pathogens, aredesired for rapid detection and diagnosis of infectious disease, forscreening for environmental and food contaminants, and for efficientbiodefense screening and response procedures (reviewed in (Iqbal, et al.(2000), Biosens Bioelectron, 15:549-78)). Flaviviruses, for example, area group of positive-stranded RNA viruses that have a global impactresulting from their widespread distribution and ability to causedisease in humans and economically important domestic animals. Severalmembers of this genus, such as dengue virus (DEN) and West Nile virus(WNV), are considered emerging or re-emerging pathogens because of therapid annual increase in the rate at which they encounter humans andcause disease (Gubler (1998), Clin Microbiol Rev, 11:480-96). With 50million cases of related illness reported annually, DEN infection hasbecome the most significant source of arthropod-borne viral disease inhumans (Gubler, et al. (1993), Infect Agents Dis, 2:383-93, Monath(1994), Proc Natl Acad Sci USA, 91:2395-400). Both DEN and WNV areemerging biodefense pathogens (category A and B, respectively) for whichthe development of diagnostics, therapeutics, and vaccines are the focusof considerable effort. The development of a DEN vaccine is particularlychallenging because sequential exposure to different serotypes of DENactually increases (rather than attenuates) the likelihood of developingdengue hemorrhagic fever in response to infection (Burke, et al. (1988),Am J Trop Med Hyg, 38:172-80, Graham, et al. (1999), Am J Trop Med Hyg,61:412-9, Sangkawibha, et al. (1984), Am J Epidemiol, 120:653-69,Vaughn, et al. (2000), J Infect Dis, 181:2-9, Winter, et al. (1968), AmJ Trop Med Hyg, 17:590-599). For such pathogens, characterizing not onlythe magnitude, but also the breadth, persistence, and specificity of thehumoral response is an important component of evaluating candidatevaccines and understanding pathogenesis in infected individuals.

Conventional laboratory techniques used to detect pathogens andantibodies in blood serum and other samples include ELISA, PCR, and cellculture (Belgrader, et al. (1999), Science, 284:449-50, Belgrader, etal. (2003), Anal Chem, 75:3114-8, Rowe, et al. (1999), Anal Chem,71:3846-52). A common limitation of these techniques is the time andcomplexity of the assays themselves, which usually require extensivesample handling. This renders them unsuitable for rapid screening ofsamples, for automation, and for portability for field applications.Recent directions in the development of alternative techniques forpathogen detection reflect these limitations in traditional technology,and the need for increased simplicity, reliability, and rapidity formethods that detect both biodefense pathogens and antibodies that targetthem.

Increasingly, biosensor systems are utilizing living cells, and theircomplex sensing and signaling mechanisms, for assays such as pathogendetection (Bechor, et al. (2002), J Biotechnol, 94:125-32, Belkin(2003), Curr Opin Microbiol, 6:206-12, Conway, et al. (2002), ReceptorsChannels, 8:331-41, Haruyama (2003), Adv Drug Deliv Rev, 55:393-401,Kamei, et al. (2003), Biotechnol Lett, 25:321-5, Karube, et al. (1994),Curr Opin Biotechnol, 5:54-9, Park, et al. (2003), Biotechnol Prog,19:243-53). Cell-based biosensors are already being exploited indiagnostic and screening tests, including some for biodefense pathogens.For example, a technique was reported (Rider, et al. (2003), Science,301:213-5) in which B-cells were used to report the presence ofclone-specific pathogens. These cells were transfected with acalcium-sensitive bioluminescent protein that was sensitive tofluctuations in cytosolic calcium resulting from B-cell receptor (BCR)signaling. Cell-based assays of this sort show improvements in fidelity,simplicity, and speed when compared with traditional pathogen detectiontechniques. However, despite their advantages compared with traditionalmethods, assays that utilize living cells are limited in three mainareas: 1) Dependence on living cells that require high maintenance andspecialized tissue culture facilities, rendering miniaturization (due tocell size and environmental requirements) and field applicationimpractical or impossible. 2) Limitation in flexibility of antigenrecognition due to the clonal nature of cell pathogen-receptors (such asBCRs), requiring extensive cell line development for detection ofdiverse pathogens or of mutants and variants. 3) Susceptibility to falsepositives resulting from reliance on a single pathogen receptor, and ondetection of a single downstream (and often promiscuous) signaling event(e.g. Ca⁺⁺ flux). There is a need for the development of pathogen (andligand) biosensors that can utilize cell-sensing and signaling machineryin a flexible and cell-free format in order to overcome theselimitations.

Sensors that take advantage of the signaling capacity ofsingle-transmembrane (1-TM) proteins, such as BCRs and kinase-activatingreceptors, have not been utilized outside of laboratory-based live-cellassays. Cells have been required for both the maintenance of the nativestructure of 1-TM proteins (which is reliant upon the presence of thecell membrane), and for the retention of cellular signaling pathwaysthat can be linked to a measurable reporter. 1-TM receptors typicallycomprise an extracellular ligand recognition domain, a transmembranedomain that crosses the cell membrane once and anchors the protein tothe cell surface, and one or more intracellular domains which interactdirectly or indirectly with cytosolic signaling proteins. Functionally,many 1-TM receptors mediate their signaling activities through a commonmechanism—ligand-induced receptor cross-linking. For example, BCRs existas unliganded monomers on the cell surface that, when cross-linked by anappropriate antigen, form clusters on the cell surface. Clustering andcross-linking of 1-TM receptors induces a cascade of phosphorylationevents that recruits adaptors and other accessory proteins to thereceptor complex, ultimately activating multiple signaling pathways suchas calcium/calmodulin, phospholipase C, Ras, and MAPK. The ability toeasily manipulate 1-TM pathogen and ligand recognition elements, andlink them to signaling and reporter (output) pathways in cell-freevehicles, could enable new types of pathogen sensors to be developed,exhibiting many of the advantages of live-cell assays, but without theirpractical limitations.

One method of measuring protein-protein interactions is by fluorescentresonance energy transfer (FRET). When complementary fluorescentreporters are brought into close proximity, the transfer of fluorescentenergy from an excited donor (CFP) to an acceptor (YFP) results influorescence emission by the acceptor (Stanley (2003), ChromaApplication Note No. 6). The transfer of energy is by non-radiativedipole-dipole interaction, making FRET efficiency highly dependent uponfluorochrome pair proximity (within 5 nm), and thus an excellentindicator of proximity. FRET strategies have been used on numerousoccasions within cells and cell-based biosensors to measure interactionsbetween membrane proteins (Chan, et al. (2001), Cytometry, 44:361-8,Minor (2003), Curr Opin Drug Discov Devel, 6:760-5, Overton, et al.(2000), Curr Biol, 10:341-4, Overton, et al. (2002), Methods, 27:324-32,Tertoolen, et al. (2001), BMC Cell Biol, 2:8). A similar system using aluminescent and fluorescent pair is also available (BRET).

Thus there is a need for improved molecules and methods for thedetection of antigens and ligands. The present invention fulfils theseneeds as well as others.

G protein coupled receptors (GPCRs) are a large family of cell surfacereceptors with an assortment of ligands and diverse biological actions.The importance of GPCRs in cellular function, their diversity, and theiraccessibility to exogenous agents make them an important focus ofresearch into disease processes and drug discovery.

GPCR activation events are communicated to cell signaling pathways viaGTP-binding proteins (G proteins) associated with the intracellulardomain of the receptor. GPCRs constitute the largest group of drugtargets today, highlighting their importance in biological research andin disease pathways. However, GPCRs are structurally complex, spanningthe cell membrane seven times. Removal from the cell membrane usuallydestroys the receptor's native structure which is maintained by theenvironment of the lipid bilayer. GPCRs are thus extremely difficult topurify and manipulate experimentally, and their study relies on wholecells or isolated cell membranes. However, these formats suffer frompoor receptor purity, stringent environmental requirements, and aninability to be miniaturized, prohibiting their application to emergingmicro- and nano-scale detection technologies. Methods for assaying GPCRactivation that can be applied to microfluidic drug-screening devicesare needed.

Although GPCRs respond to a wide variety of extracellular ligands, theymediate intracellular communication through common signaling pathways(Kiselyov, et al. (2003), Cell Signal, 15:243-53, Morris, et al. (1999),Physiol Rev, 79:1373-430). The intracellular domains of GPCRs arecoupled to a heterotrimeric complex of membrane-associated GTP-bindingproteins (G proteins). Nearly all GPCRs initiate their signaling pathwaythrough the action of G proteins, which transmit the GPCR activationsignal to intracellular effectors. The G protein complex consists of Gα,Gβ, and Gγ subunits, each of which occur as a number of ligand- andsignal-specific isotypes. For example, the Go family includes Gi, Gs,Gq, and G₁₂ isotypes, and a family such as Gi is composed of severalsub-members (Gi, Go, Gt, Ggus, and Gz). In the inactive state, the Gαsubunit binds GDP and maintains the GPCR in a ligand-receptiveconformation. GPCR stimulation by an agonist induces Gα to exchange GDPfor GTP. The now activated G protein subunits dissociate and activatesignaling cascades that release second messengers such as cAMP andintracellular calcium. These second messengers exert their biologicaleffects by modifying cellular processes such as gene expression, ionbalance, and the release of bioactive substances. The hydrolysis of GTPto GDP by Gα returns the G proteins to their inactive state, attenuatingand eventually terminating the signal. Multiple accessory proteins, suchas arrestins and GTPase-activating proteins (GAPs), modify thesedownstream signaling events. A number of studies have created GPCR-Gprotein fusion proteins (Milligan (2000), Trends Pharmacol Sci, 21:24-8,Milligan (2002), Method in Enzymology: G Protein Pathways Part A,343:260-273, Molinari, et al. (2003), J Biol Chem, 278:15778-88).

GPCR activation is traditionally measured experimentally by monitoringone or more of the participants of these signaling cascades.Fluorescently or radioactively labeled GPCRs, G-proteins, and guaninenucleotides, have all been cited as potential reporters of intracellularsignaling events (Eidne, et al. (2002), Trends Endocrinol Metab,13:415-21, Hemmila, et al. (2002), Drug Discov Today, 7:S150-6, Kimple,et al. (2001), J Biol Chem, 276:29275-81, Milligan (2003), TrendsPharmacol Sci, 24:87-90, Moore, et al. (1993), Biochemistry, 32:7451-9,Remmers (1998), Anal Biochem, 257:89-94, Remmers, et al. (1996), J BiolChem, 271:4791-7, Remmers, et al. (1994), J Biol Chem, 269:13771-8).However, ‘end-point’ messengers such as calcium flux or cAMP productionare not stimulated by a number of important GPCRs and G proteins, and assuch, the ligands and functions of many GPCRs remain unknown.

Thus there is a need for improved methods for detecting GPCR activationto overcome the current constraints by being simple, flexible, andapplicable to a wide range of GPCRs and G-proteins. The presentinvention fulfils these needs as well as others.

Proteins that span the membrane multiple times present a unique set ofchallenges for structural analyses such as x-ray crystallography. As aresult, the structure of only a handful of multiple-spanning membraneproteins has been determined at high resolution (for example, see(Jiang, et al. (2002), Nature, 417:515-22, Jiang, et al. (2003), Nature,423:33-41, Palczewski, et al. (2000), Science, 289:739-45,Pebay-Peyroula, et al. (1997), Science, 277:1676-1681) (approximately 40unique membrane protein structures compared to 3,000 unique solubleprotein structures) (Nollert, et al. (2004), DDT: Targets, 3:2-4,Werten, et al. (2002), FEBS Lett, 529:65-72), and the vast majority ofthese proteins are relatively simple, prokaryotic proteins (52 of 67total membrane protein structures are of bacterial origin) (Werten, etal. (2002), FEBS Lett, 529:65-72).

Structural studies of any protein typically require the protein to beexpressed at high levels, solubilized for manipulation, and purified tonear complete homogeneity. Integral membrane proteins presentdifficulties in all three of these requirements. While soluble (secretedor cytoplasmic) proteins can be expressed at high levels withintraditional E. coli and insect cell expression systems, membraneproteins have been less successful, in part because these systems lackmany of the (poorly understood) folding, membrane insertion, andtrafficking mechanisms used to express higher-order eukaryotic membraneproteins. Oligomeric membrane proteins present even greaterdifficulties, as co-expression of subunits and assembly systems arerequired for efficient production of structurally intact functionalunits. In addition, the membrane surface area of cells is limited,restricting the quantity of membrane protein that can be expressed inany given cell (e.g. compared to secreted and intracellular proteins).Exemplifying this limitation, rhodopsin, the sole GPCR for which thecrystal structure has been determined, was derived from natural tissue(bovine retinas) that contain unusually large amounts of the protein(Palczewski, et al. (2000), Science, 289:739-45). Comparable naturalsources for other membrane proteins are rarely available. Despite thesedifficulties, however, several membrane proteins have been successfullyexpressed at high levels, often using baculovirus insect cell expressionsystems (Klaassen, et al. (1999), Biochem J, 342 (Pt 2):293-300,Lundstrom (2003), Biochim Biophys Acta, 1610:90-6, Massotte (2003),Biochim Biophys Acta, 1610:77-89, Nollert, et al. (2004), DDT: Targets,3:2-4). Mammalian expression systems, such as semliki forest virus(SFV), have also been used to obtain large quantities of membraneproteins (Lundstrom (1997), Curr Opin Biotechnol, 8:578-82, Lundstrom(2003), Biochim Biophys Acta, 1610:90-6, Lundstrom, et al. (2001), FEBSLett, 504:99-103, Wurm, et al. (1999), Curr Opin Biotechnol, 10:156-9).Just as importantly, advances in crystallization have extended theuseful life of limited quantities of protein by miniaturizingcrystallization trials (2 mg of protein is often now sufficient for anentire trial).

Despite the successful large-scale expression of at least some membraneproteins, membrane proteins are still difficult to crystallize. Aprimary reason for this disparity is because once expressed, membraneproteins face another obstacle—purification to homogeneity. Whilesoluble proteins can be readily purified from the cellular media (in thecase of secreted proteins) or soluble cell fractions (in the case ofcytoplasmic proteins), membrane proteins remain embedded within the cellwhere they are difficult to extract. A significant portion of manymembrane protein molecules, sometimes even a majority of the protein, isembedded within the plasma membrane lipid bilayer. GPCRs for example,possess seven distinct transmembrane domains, making large portions ofthese proteins hydrophobic and the protein as a whole topologicallycomplex. Removal of GPCRs (and most other multi-spanning membraneproteins) from their lipid bilayer usually results in loss of theirnative structure. Because 95% of the cell's membrane content is insidethe cell (nucleus, mitochondria, endoplasmic reticulum, golgi, etc.),purification of plasma membrane proteins is not trivial. Detergents canallow solubilization of some membrane proteins in their native state,but this is an empirical and very time-consuming process, and even thenstability is only transient. Finding a detergent that keeps a membraneprotein structurally intact, in micelle form, and stable enough forpurification is difficult; finding one that is selective enough toaccomplish these things in the presence of a large amount ofcontaminating lipid and protein limits the choices of detergent evenfurther. Finally, even when membrane proteins can be expressed andpurified from cells using detergents, cell lysates will contain aheterogeneous mix of the membrane protein of interest in various stagesof synthesis, folding, and processing.

Therefore, there is a need for innovations that focus primarily on thedevelopment of methods for membrane protein preparation. A novel methodthat can enable the purification of large quantities of homogeneousmembrane protein could have a major impact on drug discovery andmembrane protein research.

Baculovirus vectors are commonly used to express high quantities ofmembrane proteins in their correctly folded, natively processed forms(Carfi, et al. (2002), Acta Crystallogr D Biol Crystallogr, 58:836-8,Carfi, et al. (2001), Mol Cell, 8:169-79, Klaassen, et al. (1999),Biochem J, 342 (Pt 2):293-300, Massotte (2003), Biochim Biophys Acta,1610:77-89). Baculovirus systems are limited to particular cell types(most commonly insect Sf9 or High Five cells), but result in very highlevels of expression from the polyhedrin promoter in serum-free growthconditions. Insect cell-derived proteins are routinely used incrystallography studies, a result of their combined high protein yieldand ability to produce correctly folded and processed eukaryoticproteins. Baculovirus vectors expressing Gag (from HIV and from MLVretroviruses) have previously been used to study retroviral assemblymechanisms (Adamson, et al. (2003), Virology, 314:488-96, Gheysen, etal. (1989), Cell, 59:103-12, Hughes, et al. (1993), Virology,193:242-55, Royer, et al. (1992), J Virol, 66:3230-5, Yamshchikov, etal. (1995), Virology, 214:50-8, Yao, et al. (2003), Vaccine, 21:638-43,Yao, et al. (2000), AIDS Res Hum Retroviruses, 16:227-36, Zemanova, etal. (2004), Biochemistry).

Vaccinia virus vectors are one of the most potent expression systemswithin mammalian cells. Vaccinia is capable of infecting nearly any celltype and expresses high amounts of protein driven from an internalvaccinia promoter (synthetic early-late promoter). Both transcriptionand translation of vaccinia genes occur in the infected cell'scytoplasm, enabling high level expression of nearly any protein.Vaccinia expressing Gag proteins have previously been described as ameans of studying retroviral assembly and budding (Karacostas, et al.(1989), Proc Natl Acad Sci USA, 86:8964-7). Vaccinia vectors can beeasily produced by recombination between a specially engineered plasmid(psC60) and a wild type strain of the virus (WR), followed by plaquepurification. Advantages of vaccinia also include native mammalian cellprocessing and trafficking. Replication-deficient vaccinia virus MVA canalso be used to reduce virus-induced toxicity and the presence of livevirus.

Alphaviruses, such as semliki forest virus (SFV), have proven to beamong the most robust mammalian expression systems described to date,especially for correctly folded and processed eukaryotic membraneproteins (Lundstrom (1997), Curr Opin Biotechnol, 8:578-82, Lundstrom(2003), Biochim Biophys Acta, 1610:90-6, Lundstrom, et al. (2001), FEBSLett, 504:99-103, Wurm, et al. (1999), Curr Opin Biotechnol, 10:156-9).Their utility reflects three major attributes. First, the geneticorganization of the SFV genome allows the introduction of heterologousgenes in place of genes encoding the viral structural proteins, wherethey are under the control of an internal sub-genomic promoter (26S).Second, RNAs encoded by alphavirus vectors (called replicons) arecapable of cytoplasmic replication in transduced cells. Replication ofthe replicon RNA in the cytoplasm effectively increases the number oftemplates for transcription and bypasses mRNA nuclear exportlimitations, resulting in high-level gene expression. Finally, when SFVstructural genes are provided in trans, SFV replicons can be packagedinto virus particles capable of single-round infection of virtually anycell type. SFV vectors expressing Gag (from HIV and from MLVretroviruses) have previously been used to study viral assembly and toproduce more effective vaccines (L1, et al. (1996), Proc Natl Acad SciUSA, 93:11658-63, Suomalainen, et al. (1994), J Virol, 68:4879-89,Weclewicz, et al. (1998), J Virol, 72:2832-45). While SFV is capable ofhigh levels of protein expression within hours of infection, the virusnormally kills infected cells within several days. Mutations of SFV havebeen characterized that delay or prevent cytotoxicity that can be used(Lundstrom, et al. (2001), FEBS Lett, 504:99-103). Numerous alternativealphavirus expression systems exist (pSFV-help, Invitrogen) and/or areemerging (producer cells with capsid-E1-E2/3) that continue to improvedease of use and viral titer.

Recombinant adenovirus expressing Gag has previously been developed as ameans of producing retroviruses for gene therapy (Caplen, et al. (1999),Gene Ther, 6:454-9, Duisit, et al. (1999), Human Gene Therapy,10:189-2000, Lin (1998), Gene Therapy, 9:1251-1258, Ramsey, et al.(1998), Biochem Biophys Res Commun, 246:912-9, Torrent, et al. (2000),Cancer Gene Ther, 7:1135-44, Yoshida, et al. (1997), Biochem Biophys ResCommun, 232:379-82).

SUMMARY OF THE INVENTION

The present invention provides lipoparticles comprising a viral proteincomponent and a cellular protein, wherein said viral protein componentconsists essentially of a viral structural protein.

In some embodiments, the present invention provides lipoparticlescomprising a viral protein component and a cellular protein, whereinsaid cellular protein is an unmodified protein, and wherein saidlipoparticle is reverse transcription incompetent.

The present invention also provides lipoparticles comprising anunmodified viral structural protein and a cellular protein, providedthat the only viral proteins in said lipoparticle are structuralproteins.

In some embodiments, the present invention provides lipoparticlescomprising a viral structural protein and a native cellular protein,provided that the only viral proteins in said lipoparticle arestructural proteins.

The present invention provides compositions comprising an isolatedlipoparticle of any attached to a biosensor surface.

In some embodiments, the present invention provides lipoparticlescomprising a viral protein component, a cellular protein and aG-protein.

In some embodiments, the present invention provides methods ofidentifying modulators of a GPCR comprising: a) contacting alipoparticle comprising a GPCR and a G-protein with a test compound; andb) measuring GPCR activity.

In some embodiments, the present invention provides methods forproducing a lipoparticle comprising: a) contacting a cell with nucleicacid encoding an unmodified viral structural protein and a cellularprotein; and b) culturing said cell under conditions resulting inproduction of said lipoparticle, provided that the only viral proteinencoded by said nucleic acid is a structural protein.

In some embodiments, the present invention provides methods forproducing a lipoparticle comprising: a) contacting a cell having amembrane protein of interest with an adenovirus encoding at least aviral Gag protein; and b) culturing said cell under conditions resultingin production of said lipoparticle.

In some embodiments, the present invention provides methods forproducing a lipoparticle comprising: a) contacting a cell with anadenoviral vector encoding at least a viral Gag protein and a cellularprotein or an adenoviral vector encoding at least a viral Gag proteinand a nucleic acid encoding said cellular protein; and b) culturing saidcell under conditions resulting in production of said lipoparticle.

In some embodiments, the present invention provides chimeric viralvectors comprising adenoviral nucleic acid and retroviral nucleic acid,provided that said retroviral nucleic acid comprises a sequence encodingGag, but does not comprise a sequence encoding the envelope, promoter,or packaging signal of the retrovirus.

In some embodiments, the present invention provides methods of elicitingan immune response in a subject comprising administering lipoparticlesto said subject.

The present invention also provides methods of assessing the bindinginteraction of a protein with a ligand, said method comprisingcontacting a lipoparticle comprising said protein, wherein saidlipoparticle is attached to a substrate, with a ligand of said protein;and detecting any change in said substrate compared with any change inan otherwise identical substrate wherein said lipoparticle is notcontacted with said ligand; wherein detecting a change in said substratewherein said lipoparticle is contacted with said ligand compared withsaid otherwise identical substrate wherein said lipoparticle is notcontacted with said ligand assesses said binding interaction of saidprotein with said ligand.

The present invention also provides methods of identifying potentialligands of a protein, said method comprising contacting a lipoparticlecomprising said protein, wherein said lipoparticle is attached to asubstrate, with a test ligand and detecting any change in said substratecompared with any change in an otherwise identical substrate whereinsaid lipoparticle is not contacted with said ligand; wherein detecting achange in said substrate wherein said lipoparticle is contacted withsaid ligand compared with said otherwise identical substrate whereinsaid lipoparticle is not contacted with said ligand identifies a ligand.

The present invention also provides methods of identifying a compoundthat affects binding between a ligand and a protein, said methodcomprising contacting said compound with said ligand; contacting saidcompound/ligand complex with a lipoparticle comprising said protein,wherein said lipoparticle is attached to a substrate; and detecting anychange in said substrate compared with any change in an otherwiseidentical substrate wherein said compound is not contacted with saidligand and said ligand is contacted with said lipoparticle; whereindetecting a change in said substrate when said compound is contactedwith said ligand compared with said otherwise identical substratewherein said ligand is contacted with said lipoparticle assesses saideffect of said compound.

In some embodiments, the present invention provides methods of detectinga ligand of a protein in a test sample, said method comprisingcontacting a lipoparticle comprising said protein, wherein saidlipoparticle is attached to a substrate with a test sample; anddetecting any change in said substrate compared with any change in anotherwise identical substrate wherein said lipoparticle is not contactedwith said test sample; wherein detecting a change in said substratewherein said lipoparticle is contacted with said ligand compared withsaid otherwise identical substrate wherein said lipoparticle is notcontacted with said test sample indicates the presence of said ligand insaid test sample.

In some embodiments, the present invention provides immunogenscomprising a lipoparticle.

The present invention also provides methods of eliciting an immuneresponse to a protein, said method comprising the introduction of alipoparticle to an animal.

In some embodiments, the present invention provides methods of elicitingan immune response to a protein, said method comprising the introductionof a lipoparticle to an animal.

In some embodiments, the present invention provides methods ofdetermining the structure of a membrane protein comprising: a) isolatinga membrane protein from a lipoparticle containing said membrane protein;and b) determining said structure of said membrane protein; wherein saidmembrane protein is not a viral envelope protein.

In some embodiments, the present invention provides methods ofdetermining the structure of a protein comprising: a) isolating aprotein from a lipoparticle containing said membrane protein; and b)determining said structure of said protein; wherein said proteincomprises a Gag fusion protein.

In some embodiments, the present invention provides compositionscomprising an array of lipoparticles attached to a surface.

In some embodiments, the present invention provides methods of detectingan infectious pathogen in a sample comprising the steps of: a)contacting the sample with an array of lipoparticles attached to asurface, wherein said array of lipoparticles comprises membrane proteinsthat interact with various infectious pathogens; and b) detecting aninteraction with said array of lipoparticles; wherein said detection ofsaid interaction indicates the presence of an infectious pathogen.

In some embodiments, the present invention provides methods ofdetermining the presence of a substance in a sample comprising the stepsof: a) contacting the sample with an array of lipoparticles attached toa surface, wherein said lipoparticles comprise membrane proteins thatinteract with said substance; and b) detecting an interaction with saidarray of lipoparticles; wherein said detection of said interactionindicates the presence of said substance.

In some embodiments, the present invention provides methods ofidentifying an inhibitor of a binding activity of a substance to amembrane protein comprising the steps of: a. contacting said substancewith an array of lipoparticles comprising said membrane protein attachedto a surface to which said substance normally binds, in the presence ofa potential inhibitor; and b. detecting an interaction of said substancewith said array; wherein if an interaction is detected, then saidpotential inhibitor does not inhibit said binding and if an interactionis not detected then said potential inhibitor inhibits said binding.

In some embodiments, the present invention provides methods for spottinglipoparticles, viruses, or virus-like particles in an array format ontoa surface comprising including in the spotting medium a preservative.

In some embodiments, the present invention provides methods ofidentifying a binding partner of a membrane protein comprising: a)contacting a surface coated with lipoparticles, viruses, or virus-likeparticles comprising said membrane protein with an array comprisingpotential binding partners; and b) detecting binding of potentialbinding partner to said membrane protein.

In some embodiments, the present invention provides lipoparticlescomprising a viral protein component and a cellular protein, whereinsaid viral protein component consists essentially of a viral structuralprotein, wherein said cellular protein is an ion channel protein ortransporter protein.

In some embodiments, the present invention provides methods to determinemembrane protein function in a lipoparticle, virus, or virus-likeparticle comprising a membrane protein, wherein said lipoparticle,virus, or virus-like particle further comprises a detectable agent,wherein measuring either an increase or decrease in the detectable agentis used to determine the membrane protein function.

In some embodiments, the present invention provides methods ofidentifying a stimulator of a membrane protein comprising: a) contactinga lipoparticle comprising said membrane protein and a detectable agentwith a compound; and b) measuring any change in the detectable agent;wherein said change in the detectable agent is used to indicate thatsaid compound is a stimulator.

In some embodiments, the present invention provides methods ofidentifying an inhibitor of a known stimulator of an ion channel proteinor a transporter protein within a lipoparticle, wherein saidlipoparticle comprises an ion channel or transporter, comprising thesteps of: a) contacting said lipoparticle with said stimulator; b)contacting said lipoparticle with a test compound; c) measuring thefunction of said ion channel protein or transporter protein.

In some embodiments, the present invention provides methods of detectingchanges in ion concentration in a location comprising: a) microinjectinglipoparticles comprising a membrane protein and a detectable agent tosaid location; and b) detecting changes in ion concentration bymeasuring said change in said detectable agent.

In some embodiments, the present invention provides immunogeniccompositions comprising a lipoparticle comprising a protein of interestand at least one immunostimulatory component.

In some embodiments, the present invention provides methods of producingantibodies against a protein comprising: a) administering a immunogeniccomposition comprising said protein to an animal; and b) isolating saidantibodies.

In some embodiments, the present invention provides methods ofidentifying a binding partner of a membrane protein comprising: a)contacting a lipoparticle, virus, or virus-like particle comprising saidmembrane protein with a library, wherein said library comprises morethan one potential binding partner; b) detecting the binding of saidbinding partner to said membrane protein.

In some embodiments, the present invention provides methods oftransfecting a protein into a cell comprising contacting said cell witha lipoparticle comprising said protein.

In some embodiments, the present invention provides methods oftransfecting a protein into a cell comprising contacting said cell witha lipoparticle comprising a viral protein component and said protein,wherein said viral protein component consists essentially of a viralstructural protein.

In some embodiments, the present invention provides methods ofcorrecting a protein defect in an individual comprising administering acell transfected with a lipoparticle.

In some embodiments, the present invention provides particles comprisinga fluorophore wherein said fluorophore changes fluorescence in responseto pH, membrane potential, oxidation state, NO level, ion concentration,ATP concentration, protein interaction, or combinations thereof andwherein said particle is less than 1 μm

In some embodiments, the present invention provides lipoparticlescomprising a Gag fusion protein and exogenous membrane protein, whereinsaid Gag fusion protein comprises a fluorescent protein or an enzymaticprotein.

In some embodiments, the present invention provides lipoparticlescomprising a modified lipid.

In some embodiments, the present invention provides lipoparticlescomprising at least one of a radioactive molecule, a magnetic substance,a paramagnetic substance, a biotinylated molecule, an avidin-likemolecule, gold, or combinations thereof and optionally a fluorophore.

In some embodiments, the present invention provides methods ofincorporating a molecule into a lipoparticle, virus or a virus-likeparticle comprising contacting an AM-ester form of said molecule with alipoparticle comprising an esterase.

In some embodiments, the present invention provides methods ofincorporating a molecule into a lipoparticle, virus or a virus-likeparticle comprising contacting a soluble form of said molecule with saidlipoparticle and performing electroporation, sonication, or vortexing.

In some embodiments, the present invention provides methods of inducingpores in a lipoparticle comprising incubating said lipoparticle with apore-forming peptide, an alkane, or a detergent.

In some embodiments, the present invention provides methods of attachinga molecule to a lipoparticle, virus, or virus-like particle comprisingcontacting a modified molecule with said lipoparticle, virus, orvirus-like particle, wherein said lipoparticle, virus, or virus-likeparticle is able to bind to said modified molecule.

In some embodiments, the present invention provides methods ofdetermining binding of a compound to a lipoparticle, virus, orvirus-like particle comprising a) contacting said compound with saidlipoparticle; and b) determining if said compound binds to saidlipoparticle, wherein said compound or said compound and saidlipoparticle comprises a fluorescent label.

In some embodiments, the present invention provides methods of detectingthe presence of an antigen in a sample comprising: a) contacting alipoparticle comprising a binding partner for said antigen with saidsample; and b) detecting a signal in said sample; wherein said detectionof said signal indicates the presence of said antigen.

In some embodiments, the present invention provides methods ofhybridizing an oligonucleotide to a target sequence in a lipoparticle,virus, or virus-like particle comprising contacting said oligonucleotidewith said lipoparticle, virus, or virus-like particle comprising saidtarget sequence under conditions that permit hybridization of saidoligonucleotide to said target sequence.

In some embodiments, the present invention provides methods of detectinglipoparticle fusion comprising: a) contacting a lipoparticle, virus, orvirus-like particle containing a fusigenic membrane protein with alipoparticle comprising a receptor for said fusigenic membrane protein;and b) detecting said fusion; wherein said lipoparticle, virus, orvirus-like particle comprises at least one reporter that is detectableupon fusion.

In some embodiments, the present invention provides lipoparticles,viruses, or virus-like particles attached to a bead, wherein saidlipoparticle is attached to said bead via WGA, PEI, avidin-biotininteraction, poly-lysine interaction, or covalent coupling.

In some embodiments, the present invention provides methods forcalculating the number of lipoparticles, viruses, or virus-likeparticles in a sample comprising: a) labeling said particles with afluorophore; b) detecting said labeled particles, and c) counting saidparticles.

In some embodiments, the present invention provides methods forcalculating the quantity of particles, wherein said particles arelipoparticles, viruses, or virus-like particles comprising: a) measuringa detectable properties of a particle sample; and b) determining saidquantity of particles by a correlation of amount of said properties toan amount of said particles.

In some embodiments, the present invention provides methods fordetecting the structural integrity of a membrane protein within aparticle comprising a) contacting said particle with a molecule thatbinds to said membrane protein; and b) detecting binding of saidmolecule to said particle; wherein binding of said molecule to saidparticle is indicative that the structural integrity of said membraneprotein is intact.

In some embodiments, the present invention provides methods fordetermining the purity of a particle, wherein said particle is alipoparticle, virus, or virus-like particle preparation comprising: a)quantifying number of particles in said preparation; b) quantifyingtotal protein concentration in said preparation; and c) determining saidpurity by dividing the total protein concentration by the number ofparticles; and d) dividing the number obtained from step c) by thetheoretical protein weight of said particle, wherein a value of about 1is indicative of a pure sample and a value greater than 1 is indicativeof a sample that is not completely pure.

In some embodiments, the present invention provides lipoparticlescomprising at least one fusion protein, wherein said fusion proteincomprises at least one binding domain, at least one transmembranedomain, and at least one reporter domain.

In some embodiments, the present invention provides methods of detectingthe presence of an antigen in a sample comprising contacting said samplewith at least one lipoparticle comprising a binding partner wherein saidparticle comprises at least one fusion protein comprising at least onebinding domain, at least one transmembrane domain, and at least onereporter domain and detecting the signal from said lipoparticle.

In some embodiments, the present invention provides devices comprisingat least one lipoparticle and capable of being used to perform themethod described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B: A. Lipoparticles were produced in the cell typesindicated on the x-axis, including human HEK-293T and HeLa, quail QT6,monkey Vero, hamster BHK, mouse NIH-3T3, and cat CCC cells. Samples werecollected after two days and assayed for MLV Gag, CCR5, or transfectionefficiency using a lacZ reporter transfected in parallel. B. Dot blotresults of CCR5 expression from a separate experiment with improvedsensitivity and additional transfection modalities are shown. Cells weretransfected using multiple reagents, including CaPO4, Lipofectamine2000, Effectene, Lipofectamine, and Lipofectamine Plus. Samples werealso assayed for Gag by ELISA with results similar to that in panel A(data not shown).

FIG. 2: Lipoparticles were produced in NIH-3T3 cells using an Adenovirusexpression vector. An Adenovirus vector expressing a Gag-GFP fusionprotein (Ad-Gag-GFP) was constructed using Invitrogen's Gateway kit andused to infect murine NIH-3T3 cells. Expression of Gag-GFP is confirmedby the presence of fluorescent cells 48 h post-infection (A). TheGag-GFP fusion protein has been previously constructed and tested in apcDNA3 plasmid vector, and the GFP fusion protein does not interferewith lipoparticle production. The presence of GFP enables lipoparticlesto be visualized using a 100× objective under epifluorescentillumination. The presence of lipoparticles produced from NIH-3T3 cellswas confirmed by visualizing Gag-GFP particles (B). Lipoparticleproduction was further confirmed using western blot analysis of lysate(lys) and supernatant (sup) from NIH-3T3 cells either infected or notinfected with Ad-Gag-GFP using an anti-Gag rabbit sera (C).

FIGS. 3A and B. Antibody binding to lipoparticles on a biosensor. (A)Lipoparticles containing CXCR4 were coupled to the surface of a BIACOREbiosensor C1 chip. MAb concentrations down to 20 pM were detected(3-fold serial dilutions of the conformation-sensitive 12G5 MAb). Dataare reference-subtracted (non-specific lipoparticles), and the data fita bivalent interaction model. (B) Plot of ‘apparent’ k_(on) vs k_(off)for nine different (bivalent) MAbs against CXCR4 and CCR5. Each kineticdata point is derived from a dilution series of the MAb binding tocaptured lipoparticles (similar to the left panel). Points falling onthe same diagonal line have the same K_(D). Each binding series fit abivalent binding model, and K_(D) was calculated using the ratio ofk_(off) over k_(on).

FIG. 4. Lipoparticles were lyophilized in the presence of 0-10% sucrose,trehalose, or glycerol, as indicated, and tested for binding to a CXCR4conformation-specific monoclonal antibody (447.08) or control surfaces(a non-specific MAb) by VELISA. The presence of bound Gag in thelipoparticles was detected with a rabbit anti-Gag sera and quantitatedby ELISA on an AlphaInnotech Fluorchem 8900. Non-lyophilizedlipoparticles with the same amount of additive (‘5% w/o Drying’) werealso included as a positive control. Negative controls included additiveadded after drying without additive (‘0% Dry, 5% Binding’), anon-specific antibody (‘5%, Non-specific MAb’), and no particles.Results indicate that lipoparticles lyophilized in the presence ofadditives retain the structure of the membrane protein CXCR4.

FIG. 5. Lipoparticles were lyophilized in the presence of 0% or 5%sucrose, as indicated, and analyzed using dynamic light scattering.Lipoparticles lyophilized in the absence of sucrose demonstratedmultiple high molecular weight peaks, while lipoparticles lyophilized inthe presence of sucrose demonstrated a single, monomodal peak similar tothe unlyophilized starting material.

FIG. 6. Lipoparticles were attached to a C1 biosensor chip via WGAattachment or via NeutrAvidin attachment, as indicated. NeutrAvidinattached lipopaticles were first biotinylated.

FIG. 7. An E1 chip was constructed and lipoparticles were attached to itvia hydrophobic interactions.

FIG. 8. Incorporation of ion channels. Incorporation of the ion channelsCFTR, Kv1.3, and Shaker into three different preparations oflipoparticles. Ion channels were detected by Western blot using epitopetags on the ion channels (V5 tag on CFTR and Kv1.3, and FLAG tag onShaker).

FIG. 9. Fluorescence microscopy of GFP or di-4-ANEPPS labeledLipoparticles. A. Lipoparticles containing the fusion protein CXCR4-GFPwere imaged by fluorescence microscopy. B. DI-4 ANEPPS was added tolipoparticles to visualize them. C. 0.2 micron YG Fluoresbrite beads(Polysciences, Inc.) were imaged at the same magnification as the othertwo panels for comparison.

FIG. 10. Reactivity of mice sera against native cell surface proteinsdemonstrates reactivity specific for the parental cell line. A. Serumdiluted from 1:100 to 1:1,000,000 was used to stain parental HEK-293cells. This mouse was immunized with 125 ug of CXCR4 lipoparticles. Theshaded curve indicates pre-immune sera (indistinguishable from the 10⁻⁶dilution, dashed line) B. Serum (1:100) derived from a mouse immunizedwith 300 ug CCR5 lipoparticles was used to stain different cell types(NIH-3T3, quail QT6, Hamster CHO, or human HEK-293 cells, which expresslittle or no endogenous CCR5 or CXCR4). The same Prebleed (shaded curve)and Test bleed (open curve) sera were tested against all cell types.Each flow cytometry curve was generated using 30,000 cells stained withmouse serum and PE-coupled secondary antibody. Cells were fixed in 200ul paraformaldehyde prior to flow cytometry.

FIG. 11. Lipoparticles induce a sera response against CXCR4 and CCR5.Sera (1:100) from mice injected with lipoparticles containing CXCR4 (toppanel) or CCR5 (bottom panel) were tested by flow cytometry forreactivity against the membrane proteins using NIH-3T3 stable cell linesexpressing either CXCR4 (line) or CCR5 (shaded). The lipoparticlesresulted in detectable and specific antibody responses. The twoleft-most panels show the response of control antibodies against CXCR4(447.08) and CCR5 (45523).

FIG. 12. Effect of adjuvants on immune response. Tests were conducted toascertain the effect of two adjuvants, RIBI and Titremax Gold, on theimmune response to membrane proteins within lipoparticles. Baby HamsterKidney (BHK) cells were transiently transfected with CXCR4 or pcDNA3vector (no membrane protein) using Lipofectamine 2000 with approximately60% transfection efficiency (not shown). Cells were then stained withtest bleed or pre-bleed sera (1:100) from each mouse and tested by flowcytometry. Results suggest that mice immunized with CXCR4 lipoparticlesproduced antibodies against CXCR4.

FIG. 13. Western blot analysis of sera from mice. Sera from mouse #2 (A)and mouse #1 (B) (both immunized with CXCR4-HA lipoparticles) reactedwith the HA epitope tag, but did not detect untagged CXCR4. Arrowindicates the 50 kDa CXCR4-HA band. A dimer of CXCR4-HA is detected inB. A 60 kDa background band is seen in all cell lysates. Negativecontrol cells are untransfected and do not express CXCR4 or CCR5. C. Alighter exposure of a serum reacting with the lipoparticle (LPs) Gagprotein is shown. The Gag protein is cleaved by the viral protease intoseveral subfragments.

FIG. 14. Response of mice sera to Gag. The response of mice sera to Gag,the structural protein of lipoparticles, was tested using an ELISAassay. Null lipoparticles (without membrane proteins) lysed in TritonX-100 were used as a source of Gag and captured to wells of an ELISAplate. Both pre-bleed and test bleed sera were tested for reactivityagainst Gag. All mice test sera exhibited a response to Gag. 5 of 8pre-bleed sera also exhibited a response to Gag. Since lipoparticle Gagis from Murine Leukemia Virus (MLV), these results may be explained ifmice were previously infected by naturally occurring MLV, which is quitecommon. By ELISA and western blot, mice sera exhibited no significantreactivity against 293 cell lysate (not shown).

FIG. 15A-I. A and B. Dynamic Light Scattering. Purified lipoparticleswere analyzed by dynamic light scattering using a Proterion DyanProdynamic light scattering measuring device (A). Lipoparticles in thisexperiment measured 207.4 nm in diameter with a polydispersity of 18.1%.200 nm beads were used as a control (B) and measured 235.9 nm indiameter with a polydispersity of 8.3%. It should be noted that themodeling assumptions used by the DLS software likely amplify theestimated diameter by about 25% due to the surface composition of viralparticles versus theoretical hard spheres. C. 200 nm YG Fluoresbritebeads (Polysciences) were quantified in four different ways. The beadswere quantified by converting weight to particles/ul by Polysciences(manufacturer's specification). The beads were then quantified bydynamic light scattering (left panel), imaging the fluorescent beadswith a 100× objective and a hemocytometer under epifluorescentillumination (middle panel), and by monochrometer intensity at awavelength of 540 nm excitation and 570 nm emission (right panel). Allresults were correlated with each other and are plotted against thepublished bead count from the manufacturer. D. Lipoparticles (leftpanel) and 200 nm YG Fluoresbrite beads (right panel) were counted by100× imaging with a microscope and subjected to dynamic light scatteringto obtain intensity values (counts/sec). The results from these analysesare plotted on a log scale over a wide range of values and areindicative of high correlation between the counts and the DLS intensity.The relationship between particle counts and intensity values can berelated with a simple equation, as shown. E. Purified lipoparticlescontaining the GPCRs CXCR4 and CCR5 were analyzed by western blot. TheGPCRs contained a V5 epitope tag. On the same western blot was run apurified and quantified protein standard (GFP-V5) that also containedthe same V5 epitope tag. Using the standard, we were able to estimatethe quantity of Shaker in the lipoparticle preparation. F. Lipoparticlescontaining the GPCR CXCR4 were purified using Ni⁺² beads (purityincreasing from left to right) and visualized by SDS-PAGE gel and Sypro(Molecular Probes) staining of all proteins (left panel). The filledarrow represents CXCR4 monomer and the unfilled arrow indicates CXCR4dimer. A faint ˜43 kDa band is also seen, which likely represents CXCR4without full glycosylation, as seen previously (Berson, et al. (1996),J. Virol., 70:6288-6295). The CXCR4 protein contains C-terminal V5 andHis tags, and represents 5.7-6.3% of the total protein in the lanes. Themajor ‘contaminant’ bands shown in the starting material andflow-through are Gag structural proteins (capsid, matrix, andnucleocapsid) that compose the lipoparticle internal structure. Aseparate preparation of CXCR4 lipoparticles was prepared and similarlyvisualized by Sypro (right lane) and western blot (middle lane) (rightpanel). G. A dot blot was performed by spotting dilutions of a standardprotein (purified GFP protein containing a V5 epitope tag) and dilutionsof two different lipoparticle preparations with receptors containing theidentical V5 tag. The dot blot filter was probed with an anti-V5antibody and quantitated. The results of the quantitation enablequantitation of the amount of receptor per ul of lipoparticle. H.Lipoparticles were used for ligand binding competition assays usingradiolabeled SDF1α (the cognate ligand for CXCR4). The concentration ofCXCR4 in the lipoparticles achieved a concentration of 230.2 pmol/mgprotein, much greater than typical concentrations in cells or membranevesicles. Results are representative of two similar experiments. I. Atitration curve of radiolabeled SDF1α binding to increasing amounts ofCXCR4 lipoparticles was conducted. The amount of lipoparticles requiredto achieve a half-maximal signal (EC₅₀) for the amount of radioligandadded was 0.15 ug, again demonstrating the high concentration ofstructurally intact CXCR4 in the lipoparticles. No non-specific bindingto CXCR3 was detected. Results shown were performed in duplicate and arerepresentative of two similar experiments.

FIG. 16A-B. A. VELISA Binding Detection. Lipoparticles containing eitherCCR5 or CXCR4 were used in a VELISA assay by binding lipoparticles toELISA wells coated with antibodies against either CXCR4 (top panel) orCCR5 (bottom panel). Bound lipoparticles were lysed, analyzed for Gagprotein, and quantified in relative units of chemiluminescence. B.CXCR4-containing lipoparticles were bound to an anti-CXCR4 MAb (447.12)or a non-specific anti-CCR5 MAb (45523) in the presence or absence ofvarious additives, as indicated. Most additives had little or no effecton binding, as measured by VELISA. Additives such as Triton X-100detergent, however, completely destroyed lipoparticle structure andbinding, as expected.

FIG. 17. Quantification of lipoparticles using a hydrophobicfluorophore. The indicated amounts of lipoparticles were quantifiedusing the hydrophobic dye di-4-ANEPPS, which fluoresces only when in alipid environment. Lipoparticles were bound to WGA-coated or non-coatedELISA wells with equivalent results. Fluorescence in each well wasmeasured using a microplate fluorometer. The positive control representslipoparticles added directly to the well with di-4-ANEPPS, and thenegative control represents buffer alone (no lipoparticles).

FIG. 18. Elements of a lipoparticle-based biosensor are shownschematically. Following binding of a multimeric target, thelipoparticle-incorporated sensors will cross-link. Sensor cross-linkingwill bring complementary fluorescent proteins (CFP and YFP) into closeproximity, allowing the transfer of resonant energy (FRET), and theemission of a signal that can be measured as an increase offluorescence.

FIG. 19. A. A Gag-G protein fusion protein was transfected into cells.The cell lysate (L) and cell supernatant (S) from this transfection wasrun on an SDS-PAGE gel and probed for Gag. The negative control 293Tcells indicate no background activity. The full-length and processed Gagis shown for comparison (pCGP). Three different clones of the Gag-Gprotein (Gi) fusion are shown, with each containing the Gag-G proteinfusion protein in both the lysate and the supernatant (i.e. inlipoparticles). B. Purified lipoparticles containing a Gag-G proteinfusion protein were analyzed by western blot by probing using ananti-Gag antibody (left panel) or an anti-G protein (Gi) antibody (rightpanel). Results indicate that purified lipopaticles produced using theGag-G protein fusion protein incorporate G protein. Lipoparticles madewith Gag-Pol and with Gag-GFP are shown for comparison.

FIG. 20. Fluorescent Lipoparticles visualized by epifluorescencemicroscopy, 100× magnification. Lipoparticles were imaged forfluorescence using different fluorescent proteins (GFP, YFP, CFP) ordyes (octadecyl-rhodamine, phosphatidyl choline-NDB, diI, Di-4-ANEPPES),and using three different strategies (Gag-fusion protein, lipophilic dyeincorporation, or membrane protein-fusion proteins). 200 nm Fluoresbritebeads and nonfluorescent particles (green channel shown, red channel wassimilarly blank) are shown as controls., All images were acquired withan epifluorescent Nikon microscope with a 100× lens.

FIG. 21. Biotin can be incorporated into Lipoparticles. Lipoparticlescontaining the modified membrane lipid, biotin-phosphatidylethanolamine(biotin-PE), were created. Biotin-PE (Avanti Polar Lipids) was added topurified lipoparticles, and allowed to partition into the membrane.(Left Panel). Biotinylated lipoparticles were attached to ahigh-capacity protein-binding surface and probed with fluorescentlylabeled streptavidin. Binding of a constant amount of fluorescentlylabeled streptavidin (Molecular Probes) was dependent upon theconcentration of biotin-PE used in lipoparticle preparation, and wassaturable. The binding curve is also depicted on a linear scale (inset).(Right Panel) Binding by Neutravidin-coated ELISA plate wells (Pierce)of biotinylated lipoparticles prepared using a range ofbiotin-PE:lipoparticle ratios was measured by virus capture ELISA(VELISA) in which Gag from captured lipoparticles is measured. Values onthe x-axis are in thousands. Binding reached a maximum at abiotin-PE:lipoparticle ratio of 100,000. The decrease in binding athigher ratios is presumably due to competition for Neutravidin bindingsites by excess, unincorporated biotin-PE.

FIG. 22. Visualization of biotinylated lipoparticles. Lipoparticles wereadsorbed to a microscope coverslip, blocked with 3% BSA, and exposed toa 50 nM solution of Alexa-Streptavidin (Molecular Probes) in a 100 μlvolume for 20 minutes before the surface was rinsed three times with 10mM Hepes pH 7 100 mM NaCl and imaged. Both chemically and lipid(DPPE-biotin) biotinylated lipoparticles produced punctuate fluorescentimages through Alexa-Streptavidin binding, whereas non-biotinylatedlipoparticles did not bind the fluorescent protein. The same coverslipsurface exposed to lipid biotin alone did not demonstrate anystreptavidin binding.

FIG. 23. Functional enzymes can be incorporated into Lipoparticles. Theluciferase reporter enzyme (60 kDa) was included in lipoparticles byusing a Gag-luciferase fusion protein for particle production. Detectionof concentration-dependent luciferase activity (Promega Steady GloLuciferase Assay System) in lysed particles demonstrates retention offunction by the incorporated enzyme. Luciferase activity of conventionallipoparticles are shown as a negative control.

FIG. 24. Melittin poration of Lipoparticles. Lipoparticles wereconstructed to contain a luciferase enzyme inside the lipoparticle. Uponaddition of increasing amounts of melittin peptide, the lipoparticleswere permeabilized and luciferase substrate was allowed to enter andproduce a signal. To insure the integrity of the luciferase, after eachaddition of melittin, the same sample was lysed with Triton X-100(circles) and again measured. The results indicate that all samplescontain luciferase, but only sample with sufficient pores induced bymellitin were able to react with the luciferase substrates. Luciferaseactivity was measured using a Wallac Victor2V and mixing lipoparticleswith the Promega Steady Glo Luciferase Assay System.

FIG. 25. Affinity binding of Lipoparticles. Lipoparticles, deriveddirectly from the cell surface, naturally possess binding qualitiessimilar to cellular membranes. Cell membranes, for example, are known toadhere to wheat germ agglutinin (WGA), a lectin that binds the abundantcell surface carbohydrate, N-acetylglucosamine. Fluorescentlipoparticles (constructed using Gag-GFP) were exposed to WGA agarosebeads and control Protein A (ProA) agarose beads. Fluorescentlipoparticles were observed by microscopy to bind the WGA beads [A], butnot the ProA beads [B], demonstrating their ability to label targets ina specific manner.

FIG. 26. A transmembrane-anchored form of the ZZ binding domain fromProtein A (Tva-ZZ) was incorporated into lipoparticles made using aGag-GFP core. The lipoparticles were then exposed to a primary antibodythat could bind the ZZ domain (or none). All lipoparticles were thenexposed to a red secondary fluorescent antibody (Goat antibody notcapable of binding ZZ directly). The presence of lipoparticles in allpanels is shown in the top row, imaging for the Gag-GFP using the greenchannel. The lipoparticles containing ZZ-TM were capable of binding twodifferent antibodies, as shown by imaging of the Cy3-labeled antibody inthe red channel (bottom row, two right panels). Lipoparticles notcontaining ZZ-TM or not exposed to the antibody did not show anyfluorescence in the red channel (bottom row, left two panels).

FIG. 27. Lipoparticles can act as Specific Probes UsingMembrane-embedded Proteins. Lipoparticles can be targeted to desiredlocations using the characteristics of membrane proteins embedded intheir surfaces. Fluorescent lipoparticles (Gag-GFP), containing theseven-transmembrane protein CXCR4, were used to probe ProA beads bearinga variety of antibodies. Beads (3-10 μm) were visualized usingfluorescent light (top panels) or white light (bottom panels) and a 10×objective. CXCR4-containing lipoparticles were bound by anti-CXCR4 ProAbeads, but not by ProA beads bearing an irrelevant antibody (anti-FLAG),or by ProA beads bearing no antibody, demonstrating the specificity ofbinding by the lipoparticle membrane-embedded protein.

FIG. 28. Visualization of lipoparticles stained with a nucleic acid dye.Lipoparticles adsorbed to a microscope coverslip, blocked with BSA, andexposed to a 20 mM solution of Alexa-Streptavidin (Molecular Probes)before the surface was rinsed with HBS and imaged. Both chemically andlipid (DPPE-biotin) biotinylated lipoparticles produced punctuatefluorescent images through Alexa-Streptavidin binding, whereasnon-biotinylated lipoparticles did not bind the fluorescent protein. Thesame coverslip surface exposed to lipid biotin alone did not demonstrateany streptavidin binding.

FIG. 29. Lipoparticles can be analyzed by flow cytometry. Twopopulations of lipoparticles, one incorporating fluorescently taggedCXCR4 (CXCR4-GFP), and the other incorporating control CXCR4 (aconstruct called Lestr-HA) were simultaneously analyzed by flowcytometry. Particles were gated using forward scatter, and fluorescencewas detected at 488 nm excitation and 530 nm emission. Fluorescentlylabeled lipoparticles were characterized by higher fluorescenceintensity compared with control non-fluorescent particles, demonstratingthat labeled lipoparticles can be specifically identified using flowcytometry.

FIG. 30. Lipoparticles can be spotted on a microarray. Lipoparticleswere constructed with either a fluorescent core (Gag-GFP), a fluorescentmembrane protein (CXCR4-GFP), or non-fluorescent versions of the same(Gag and CXCR4). Sucrose was added to lipoparticles to a finalconcentration of 5%. Spots of lipoparticles were then arrayed on apolylysine slide using a microarray device. The lipoparticles wereallowed to dry and were stored at 4 C. until further use. Fluorescencein the green channel was visualized by imaging the lipoparticles with aFITC filter on an AlphaArray 7500i (top panel). The slide was thenprobed with a conformation-dependent anti-CXCR4 MAb (447.08). Binding ofthis primary antibody was detected using a Cy3-labeled secondaryantibody (red channel). After staining, the array was again visualizedusing both green and red filters (middle and bottom panels). Green spots(middle panel) indicated that the lipoparticles were still bound to theslide, and red spots (bottom panel) indicated that CXCR4 within thelipoparticles was present and structurally intact. Control Gag particleswithout CXCR4 or GFP demonstrated little or no background.

DETAILED DESCRIPTION OF THE INVENTION

The methods, modifications, and compositions disclosed herein can beapplied to or incorporated into lipoparticles, viruses (e.g.non-enveloped (e.g. adenovirus) and/or enveloped viruses (e.g.influenza)), virus-like particles, and the like.

The lipoparticle is based on retrovirus structures and enablesstructurally intact cellular proteins to be purified away from the cell.Briefly, when a retrovirus is produced from a cell, the protein core ofthe virus buds through the membrane of the cell. As a consequence, thevirus becomes enwrapped by the cellular membrane. Once the membrane‘pinches’ off, the virus particle is free to diffuse. Normally, thevirus also produces its own membrane protein (Envelope) that isexpressed on the cell surface and that becomes incorporated into thevirus. However, if the gene for the viral membrane protein is deleted,virus assembly and budding can still occur. Under these conditions, themembrane enwrapping the virus contains a number of cellular proteins.

In a 1997 manuscript published in Science, the incorporation of membraneproteins (the GPCRs CCR5 and CXCR4) into retroviral pseudotype particles(Endres, et al. (1997), Science, 278:1462-1464) was demonstrated. Afollow-up paper in the Journal of Virology demonstrated that a morecomplex receptor, an amino acid transporter that spans the membranefourteen times (MCAT-1), could also be incorporated into retroviralpseudotypes and retain its structural and functional integrity (Balliet,et al. (1998), J. Virol., 72:671-676). In both cases, the structuralintegrity of the membrane proteins was verified using functional assays(viral fusion) and conformationally-sensitive antibodies.

In a 2000 paper published in PNAS, it was demonstrated that a number ofcomplex proteins, including GPCRs, can be incorporated intolipoparticles and attached to the BIACORE biosensor (Hoffman, et al.(2000), Proc. Natl. Acad. Sci. USA, 97:11215-11220). Binding ofantibodies, peptides, and proteins to these receptors exhibitedappropriate specificity, and structural integrity of the receptors wasmaintained. An affinity of interaction between a protein and itsreceptor (HIV gp120 and CXCR4) of approximately 500 nM was alsomeasured. This affinity is sufficiently weak so as to render previousstandard binding assays unable to detect the interaction (Doranz, et al.(1999), J. Virol., 73:10346-10358, Doranz, et al. (1999), J. Virol.,73:2752-2761). The affinity with which different HIV-1 gp120 proteinsbind to chemokine receptors can be an important determinant of viralpathogenesis, so measuring these interactions has enabled practitionersto begin correlating binding affinity with disease progression. The useof the lipoparticle made this possible, and provides a clear examplewhere use of this novel approach has proven its worth. Work with theGPCRs CCR5 and CXCR4 has, to date, been directly applicable to othermembrane proteins. Over twenty different membrane proteins have beenincorporated into virus-based lipoparticles, including GPCRs, ionchannels, transporters, and Type I and Type II single transmembraneproteins.

Lipoparticles were purified using sucrose cushions, as describedpreviously (Balliet, et al. (1998), J. Virol., 72:671-676, Endres, etal. (1997), Science, 278:1462-1464, Hoffman, et al. (2000), Proc. Natl.Acad. Sci. USA, 97:11215-11220). Lipoparticles can also be purifiedusing a number of methods that are often used to purify retroviruses(Arthur, et al. (1998), AIDS Res Human Retroviruses, 3:S311-9, Ausubel,et al. (2001), Current Protocols in Molecular Biology, Dettenhofer, etal. (1999), J Virol, 73:1460-7, Le Doux, et al. (2001), Hum Gene Ther,12:1611-21, O'Neil, et al. (1993), Biotechnology (N Y), 11:173-8, Pham,et al. (2001), J Gene Med, 3:188-94, Prior, et al. (1995), BioPharm,25-35, Prior, et al. (1996), BioPharm, 22-34, Richieri, et al. (1998),Vaccine, 16:119-129, Yamada, et al. (2003), Biotechniques, 34:1074-8,1080).

In some embodiments, unwanted proteins from the cell surface are alsopresent on the surface of lipoparticles, but these can be minimized byover-expression of the receptor of interest and by the use of non-humancell types for production. It should be noted that traditional sourcesof membrane proteins—cells and membrane vesicles—also have heterogeneousprotein populations, and that lipoparticles have a much greater densityof conformationally intact receptor.

The production of lipoparticles comprising cellular virus receptors andmembrane spanning proteins using enveloped viruses is described in US2002/0183247A1, the entire contents of which is hereby incorporated byreference. Lipoparticles can also be produced using non-envelopedviruses as described herein.

The production of lipoparticles, however, has been limited by, forexample, inefficient cellular transduction systems, bottlenecks in virusproduction within a cell, premature cleavage of viral polyproteins byoverexpression of retroviral protease, and inefficient cell growthchambers dictated by cellular transfection techniques. The presentinvention provides systems and methods that overcome these difficulties.

In some embodiments of the present invention, a “chimeric” viral genomeis constructed that contains nucleic acid sequences from a virus whosestructural protein forms the core of a lipoparticle. In one embodiment,the virus structural protein is the Gag polyprotein of a retrovirus suchas, but not limited to, murine leukemia virus, rous sarcoma virus, HIV,SIV, avian leukemia virus, equine anemia virus, and the like.

This chimeric virus may be used to transduce or transfect a producerhost cell. The producer host cell may contain a membrane protein ofinterest that is naturally expressed on its cell surface. Alternatively,the producer host cell may be induced or treated in such a way as toexpress a membrane protein of interest on its cell surface.Alternatively, the producer host cell may be transduced or transfected,for example by plasmid transfection or by viral vector infection, inorder to express a membrane protein on its cell surface. The result ofa) the structural proteins of the lipoparticle, and b) the membraneprotein of interest being expressed on the cell surface, enableslipoparticles to be formed that comprise a viral core protein and themembrane protein of interest. The resulting lipoparticle need notcontain reverse transcriptase, integrase, an expressed cellulartransgene, a genomic packaging signal (psi), or a viral genome, althoughsuch constituents in some embodiments can be present. The resultinglipoparticle need not be infectious, capable of entering a cell, capableof replicating, capable of expressing a gene, capable of reversetranscription, or capable of integrating in a host's genome, although itmay possess such functions.

The invention relates to a lipoparticle which comprises a membraneprotein. The lipoparticle allows presentation of a membrane proteinwhile preserving the membrane protein's biological structure, such thatthe interaction of the protein with its cognate ligand can be studied.In certain embodiments, the lipoparticle comprises a plurality ofmembrane proteins.

The invention also relates to methods of making the lipoparticle of theinvention including, but not limited to, a method involving using aviral vector such as, but not limited to, adenovirus or Semliki forestvirus, to express the structural proteins at high levels within adesired producer cell.

The invention also relates to methods of making the lipoparticle of theinvention including, but not limited to, a method involving using aviral vector such as, but not limited to, adenovirus or Semliki forestvirus, to express the structural proteins of the virus and a separatevector, such as, but not limited to, adenovirus, alphavirus (e.g.Semliki forest virus) or a plasmid, to express the desired membraneand/or cellular protein at high levels within a desired producer cell.The invention also relates to methods of making the lipoparticle of theinvention to a method involving using a viral vector such as, but notlimited to, adenovirus or Semliki forest virus, to express both thestructural proteins of the virus and the desired membrane and/orcellular protein at high levels within a desired producer cell.

The invention also relates to methods of using the lipoparticle of theinvention to induce an immune response, with the immune response beingused to derive monoclonal antibodies or membrane protein-specificantisera. In some embodiments the immune response may be protectiveagainst infectious diseases, against autoimmune diseases, or againstcancer, by eliciting a humoral (antibody) response or a T-cell (CTL)response.

The invention also relates to methods of using the lipoparticle of theinvention to assess protein-protein binding interactions using methodssuch as, but not limited to, microfluidics-based assays or biosensors.

Composition

A “lipoparticle,” as that term is used herein, means a small particle ofabout ten nanometers to about one micrometer, comprising an externallipid bilayer further comprising a protein. The lipoparticle may also beabout ten nm to about 500 nm, about 100 to about 500 nm, about 200 toabout 400 nm, about 300 to about 399 nm, about 500 nm to about 1000 nm,about 600 to about 900 nm, or about 700 to about 800 nm. Thelipoparticle does not encompass cell membrane vesicles, which aretypically produced using empirical methods and which are usuallyheterogeneous in size. The lipoparticle also does not encompassliposomes, which typically lack core proteins that induce theirformation. In some embodiments, the lipoparticle is dense, spherical,and/or homogeneous in size.

The core, or interior, of the lipoparticle is not a crucial feature ofthe invention and can comprise any viral structural protein.Lipoparticles of the invention can be made using, without limitationthereto, a virus (e.g. a retrovirus (e.g., HIV, MLV, RSV, and the like),a vesicular stomatitis virus, and the like. The core protein can bederived from any number of viral or synthetic sources. In the preferredembodiment, the core consists of a viral structural protein that issufficient to mediate budding of a virus-like particle from the cell,taking a small piece of membrane and the proteins within it with theparticles. In some embodiments the core protein is Gag or functionalfragment thereof. In some embodiments, Gag does not comprise aheterologous tag. In some embodiments, Gag does not bind to the membraneprotein that is incorporated into the lipoparticle. In some embodiments,the membrane protein that is incorporated into the lipoparticle does nothave a heterologous tag. In some embodiments, the membrane protein thatis incorporated into the lipoparticle does not bind to Gag.

A functional fragment of Gag is any fragment of Gag that is sufficientto produce a lipoparticle when expressed in a producer cell.

Enveloped virus particles can be produced that are missing one or moreof the ordinary components of such particles, such as a portion of thegenome of the enveloped virus (Volt et al., 1977, Annu. Rev. Genet.11:203-238; Hanafusa, 1977, In: Comprehensive Virology, vol. 10,Fraenkel-Conrat et al., eds., Plenum Press, New York, pp. 401-483). Suchvirus particles are referred to herein as ‘defective.’ Lipoparticlescomprising such a defective particle and a membrane protein are includedin the present invention. It is contemplated that the omission of one ormore components of such particles provides an opportunity to substitutean additional component in place of the missing component. In addition,numerous viruses known in the art are able to accommodate the presenceof an additional component without deletion of a component of the virus.By way of example, the additional component may be a nucleic acid, anantisense nucleic acid, a gene, a protein, a peptide, Vpr protein, anenzyme, an intracellular antagonist of HIV, a radionucleotide, acytotoxic compound, an antiviral agent, an imaging agent, and the like.

In another embodiment, the defective virus facilitates the production ofthe lipoparticle. For example, the retroviral gag gene is necessary andsufficient for budding of a retrovirus-like particle. The retroviralgenes pol (including protease) and env, the packaging signal psi, andthe long terminal repeats (LTRs) need not be present for production of alipoparticle. In some embodiments, these sequences are not present.Indeed, the elimination of the protease protein (part of the pol gene)can enhance retroviral budding by avoiding premature cleavage of Gagwhen the protease is overexpressed. Similarly, substitution of the viralLTR promoter with a stronger promoter can enhance production.Elimination of all of these genes and the packaging signal (psi) makesproduction of replication competent, fusigenic, or infectious particleshighly unlikely.

Thus, the lipoparticles may comprise a simple membrane in which aprotein of interest can be embedded while maintaining the normalstructure, function, or both, of the protein. However, in someembodiments, the membrane protein may not retain a detectable functionin a lipoparticle since this is partly determined by intracellularpathways that may or may not be present inside the lipoparticle.However, in some embodiments, the membrane protein maintains itsstructure compared with the native protein when present in the membraneof a cell.

The lipoparticle may comprise a single membrane protein or a pluralityof membrane proteins. By way of example, a lipoparticle may comprise amembrane protein that is CD4, CCR5, CXCR4, ICAM-1, ICAM-2, ICAM-3, CR3,CR4, CD43, CD44, CD46, CD55, CD59, CD63, CD71, a chemokine receptor,Tva, and MCAT-1. In some embodiments, the viral vector comprises Tva,MCAT-1, CD4, CCR5, CXCR4, both CD4 and CCR5, or both CD4 and CXCR4.However, the invention is not limited to these molecules. Indeed, thedata disclosed in Balliet, et al. (Balliet, et al. (1998), J. Virol.,72:671-676) demonstrated the successful incorporation of Tva and MCAT-1into MLV (murine leukemia virus) lipoparticles, which demonstrated thattype 1 (i.e., single-spanning proteins) and an amino acid transporter(fourteen transmembrane domains), respectively, and not just multiplemembrane spanning proteins such as GCPRs, can be embedded in thelipoparticles while preserving their native binding ability.Preservation of binding ability relative to the protein as typicallypresent in the cell membrane can be assessed by functional assays, suchas, but not limited to, the biosensor assays exemplified herein.

Although the Examples described herein disclose viral vectors thatcomprise one or two membrane proteins, one skilled in the art is enabledby the teaching provided herein to produce a viral vector comprising anynumber of membrane proteins in the lipoparticle. Indeed, one skilled inthe art, based on the disclosure provided herein, would appreciate thatthe invention encompasses any membrane protein and any protein typicallypresent in a membrane can be inserted into the lipoparticles of theinvention thereby presenting the protein in its native conformationand/or preserving its binding affinity of a cognate ligand or bindingpartner. In some embodiments, a protein that is not normally targeted tothe membrane can also be incorporated into the membrane of thelipoparticle. This may be done by having a fusion protein that comprisesa protein of interest and a signal peptide that directs it to themembrane. In some embodiments, a signal peptide is removed, which allowsthe protein to be targeted to the plasma membrane. In some embodiments,the protein will be targeted to be a membrane protein. As used herein,the phrase “targeted to be a transmembrane protein” refers to a proteinthat is not normally a membrane protein but is then modified so that isbecomes a membrane protein. In some embodiments, the protein is targetedso that it attaches to the membrane but does not traverse the membrane.In some embodiments, the protein is targeted to and attached to theextracellular side of the plasma membrane but does not span themembrane.

In addition, the inclusion of two or more proteins that form a complex,quaternary structure (e.g. homo- or hetero-oligomers) can be useful fordrug discovery targeting or antibody production. In some embodiments,the protein is associated with the membrane either through non-covalentinteractions with the membrane itself or through an interaction withanother protein that is attached to the membrane.

In some embodiments, a lipoparticle comprises a multiple membranespanning protein that spans the lipid bilayer at least twice. In someembodiments, the lipoparticle comprises a membrane protein that spansthe membrane at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, or at least 14 times. The skilled artisan would understand, basedupon the disclosure provided herein, that the invention encompasses aplethora of such complex proteins which traverse a membrane multipletimes. Further, the invention provides a novel system comprising a lipidbilayer where one or more such proteins can interact to form, e.g., homoand/or heterodimers, or otherwise interact with other membrane proteinsor itself similarly to such interactions in the native membrane wherethe proteins typically reside.

Multiple membrane spanning proteins include, but are not limited to, Gprotein-coupled receptors (GPCRs) (also known as 7-transmembranereceptors, 7TM) that span the membrane seven times, e.g., CCR5, CXCR4,CCR3, mu-opioid receptor, as well as transporters (proteins thattransport molecules such as, but not limited to, amino acids orcarbohydrates, across a membrane), ion channels, and the like.

As noted elsewhere herein, the invention encompasses a lipoparticlecomprising a variety of membrane proteins, including combinations ofproteins, which can form complexes when present in the lipoparticle.Such complexes include, but are not limited to, complexes of proteinsthat function together, e.g., CD4 and CCR5, CD4 and CXCR4, and the like.The lipoparticle can comprise MCAT-1, an amino acid transporter thatspans the membrane 14 times, and ion channels that span the membrane 6times, e.g., K-channel KCNH2. All of these proteins have a similarfeature: they span the membrane at least once. Many of these span themembrane multiple times.

In addition, some complexes (e.g. homo and hetero-oligomers) interactonly when in a lipid membrane and so retain their quaternary structure(formed by multiple subunits or proteins) only when in a lipid membrane.The lipoparticle of the invention encompasses incorporation of theseproteins as well.

The membrane protein within the lipoparticle can be one which is cognateto a viral envelope protein which is displayed on the surface of a cellwith which it is desired to fuse the lipoparticle, in which case themembrane protein is defined as a cellular virus receptor. The membraneprotein may be any protein which is cognate to a viral envelope protein.Preferably, the membrane protein is cognate to a retroviral envelopeprotein, more preferably, it is cognate to a viral envelope protein of avirus selected from the group consisting of HIV, SIV, RSV, and ecotropicMLV. In some embodiments, the membrane protein is, but no limited to,CD4, CCR5, CXCR4, ICAM-1, ICAM-2, ICAM-3, CR3, CR4, CD43, CD44, CD46,CD55, CD59, CD63, CD71, a chemokine receptor, Tva, and MCAT-1. In someembodiments, the first virus receptor protein is selected from the groupconsisting of CD4, CCR5, CXCR4, Tva, and MCAT-1.

In addition to these membrane proteins, which have been exemplifiedherein, other protein types and categories are encompassed in theinvention and include, but are not limited to, the following:cytoplasmic domains of proteins (e.g., active conformations, G-proteincoupling domains, kinase motifs of proteins such as EGF-receptor, andthe like); organelle proteins (e.g., nuclear transporters, mitochondrialreceptors, endoplasmic reticulum and Golgi membrane proteins); andmultimeric complexes (e.g., dimers and trimers, viral envelope proteins,hetero-oligomers, and the like). More specifically, membrane proteins ofthe invention include, but are not limited to, GPCRs (e.g., CCR8, XCR1,CX3CR1), transporters (e.g., glucose transporter), ion channels (e.g.,K-channel Kv1.3 tetramers), tetrameric Type II protein (e.g., DC-SIGNtetramers), constitutively active GPCRs (e.g., HHV8 ORF74), viralproteins (e.g., HIV gp160, hepatitis C E1-E2 Envelope protein, expressedon endoplasmic reticulum membrane).

The lipoparticle can comprise non-membrane proteins. In someembodiments, a lipoparticle can comprise water soluble proteins thatinteract with a membrane receptor of interest. For example, alipoparticle comprising a GCPR can be made with or without G-proteins,the intracellular subunits (alpha, beta, gamma) that couple to thereceptor and mediate signaling. Other intracellular proteins are alsoincluded, such as Lck (interacts with CD4), arrestins, and β-adrenergicreceptor kinase. These intracellular proteins can influenceextracellular protein structure and can be important for formation oflipoparticles comprising complex membrane proteins that interact withsoluble intracellular proteins. These proteins can be incorporated intolipoparticles either in their native form or by targeting them forlipoparticle incorporation by e.g. inclusion of signal sequences orlipid moiety tags.

In some embodiments a lipoparticle comprises a membrane protein and acellular protein that binds to the membrane protein. In someembodiments, the cellular protein is a G-protein. In some embodimentsthe membrane protein is a GPCR. In some embodiments, the G-protein isGα, Gβ, or Gγ. In some embodiments, the G-protein is from the Gα_(i)family (Gα_(t), Gα_(gust), Gα_(i1),), Gα_(i2) family (Gα_(i3), Gα₀₁,Gα₀₂, Gα_(z)), Gα_(s) family (Gα_(s), Gα_(olf)), Gα_(q) family (Gα_(q),Gα₁₁, Gα₁₄,), Gα_(15/16), or Gα_(12/13) family (Gα₁₂, Gα₁₃). In someembodiments, the G-protein is Gβ₁, Gβ₂, Gβ₃, Gβ₄, or Gβ₅. In someembodiments, the G-protein is Gγ₁, Gγ₂, Gγ₃, Gβ₈, or Gγ_(8olf)/Gγ₉.

The membrane protein that is incorporated into the lipoparticle can be amodified membrane protein. As used herein the term “modified membraneprotein” refers to a protein that contains a portion of sequence that isnot normally found in the unmodified membrane protein. In someembodiments, the portion is at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30, at least 40, at least 50 amino acidresidues in length. In some embodiments, the portion is contiguous. Insome embodiments, the modified membrane protein comprises a fluorescentprotein. In some embodiments, the modified membrane protein comprisesCFP, YFP, GFP, eGFP, BFP, CFP, dsRED or a combination thereof.

In some embodiments, the modified GPCR comprises a fluorescent proteinin an intracellular loop of the G-protein. The structure of7-transmembrane proteins is well known to one of ordinary skill in theart and the intracellular loops and the sequences comprising thecytoplasmic tail can be identified by analyzing the sequence. In someembodiments, the fluorescent protein is present in the thirdintracellular loop, at the cytoplasmic tail, or combinations thereof.

A GPCR protein comprising a fluorescent protein at the thirdintracellular loop and the cytoplasmic tail can be used to measure GPCRactivation. When a ligand binds to a GPCR the cytoplasmic tail changesin spatial relationship to the intracellular loops and when thesecomponents comprise fluorescent proteins a change fluorescent energytransfer can be measured, which indicates that the GPCR is activated.

In some embodiments, the G-protein is a modified G-protein. A “modifiedG-protein” is a G-protein that comprises a portion of amino acidsequence that is not found in the unmodified or native G-proteinsequence. In some embodiments, the modified G-protein comprises a fusionprotein, a fluorescent protein, a linker, or a protease cleavagesequence.

The fusion G-protein can be a combination of the G-protein with aportion of another non-G-protein sequence. This fusion protein can, insome embodiments, assist in the incorporation or localization of theG-protein into the lipoparticle or to the cellular membrane of thelipoparticle. In some embodiments, the portion is a fragment or theentire sequence of another non-G-protein sequence. In some embodiments,the fusion-G-protein comprises Gag. The fusion protein can also comprisea membrane protein in addition to the G-protein. In some embodiments,the membrane protein is a single transmembrane protein. In someembodiments, the single transmembrane protein is CD4. The G-protein canalso be fused to the GPCR itself. A G-protein can also comprise afluorescent protein. In some embodiments, the G-protein comprises afluorescent protein on its N-terminus, C-terminus, or both. Thefluorescent protein of the G-protein can interact with a fluorescentfusion GPCR. The interaction of the two fluorescent proteins can bemeasured and a change in fluorescence can be used to measure GPCRactivity.

Any of these fusion proteins can also comprise a linker sequence or acleavage site. In some embodiments, the linker sequence comprises atleast 5, at least 10, at least 15, or at least 20 residues. In someembodiments, the linker sequence comprises a poly-alanine sequence, analpha helical structure, a beta-turn structure, or a beta sheetstructure. The poly-alanine sequence can comprise about 5, about 10,about 15, or about 20 alanines. The linker sequence can provideflexibility between the two proteins allowing freedom of movement andfunctionality.

The cleavage site can be any site recognized by a cellular ornon-cellular protease. In some embodiments, the protease is a viral(e.g. retroviral) protease. In some embodiments, the protease cleavagesite comprises SAAWP (SEQ ID NO:1), TAAYP (SEQ ID NO:2), SAAFP (SEQ IDNO:3), SVAYP (SEQ ID NO:4), SAGYP (SEQ ID NO:5), DDDKX (SEQ ID NO:6) orEEEKX (SEQ ID NO:7) and the like. In some embodiments, the lipoparticlecomprises the protease that is able to cleave the fusion proteincomprising the cleavage site. Examples of proteases include retroviralproteases, such as for example, HIV SIV, MLV, RSV, and ALV proteases,enterokinase. Other kinases can be found on the world wide web atdelphi.phys.univ-tours.fr/Pro-lysis/ec.html and on the world wide web atneuro.wusthedu/neuromuscular/mother/degrade.htm.

In some embodiments, a lipoparticle comprising a GPCR and a G-proteinfurther comprises a dye. The dye can also be used to measure GPCRactivity. In some embodiments, the dye is voltage sensitive or pHsensitive. Examples of dyes that can be used are described herein.

In some embodiments, the lipoparticles of the present invention comprisea GTP analog. A GTP analog can either be hydrolysable ornon-hydrolysable. In some embodiments the GTP analog is a fluorescentanalog. A fluorescent GTP analog can be used to measure GPCR orG-protein activity by measuring a change in fluorescent that occurs whenthe analog either binds to a G-protein or is cleaved by the G-protein.Examples of GTP analogs include, but are not limited to, GTPγS,MANT-GTP, MANT-GMPPNP, BODIPY-FL-GTP, BODIPY-R6G-GTP, BODIPY-TR-GTP,BODIPY FL GMPPNP, BODIPY FL GTP-γ-S thioester, TNP-GTP (2′-(or3′-)O-(trinitrophenyl)guanosine 5′-triphosphate), BzBzGTP (2′-(or3′-)O-(4-benzoylbenzoyl)guanosine 5′-triphosphate), S-(DMNPE-caged)GTP-γ-S, or Europium-GTPγS.

The G-proteins can be added to the lipoparticles in any manner thatallows the G-protein to interact with a GPCR. In some embodiments, thisincludes, but is not limited to, overexpressing the G-protein in a cellthat produces lipoparticles. The production of lipoparticles isdescribed herein. G-proteins can be overexpressed in a cell by, but notlimited to, transfection, either transiently or stably.

G-proteins can also be added to a lipoparticle by contacting thelipoparticle with purified G-protein. In some embodiments, holes orpores are made in the lipoparticle which allows the G-protein to enterthe lipoparticle. In some embodiments, holes or pores are made byelectroporation. Lipoparticles can also be permeabilized by usingmethods that involve ATP, EDTA, Ca++, Ca₃(PO₄)₂, DEAE-dextran,polyethylene-glycol, I-14402 (a cell-loading reagent), S. aureusalpha-toxin, melittin, or streptolysin-O. In some embodiments,lipoparticles are permeabilized by agitation, vortexing, or sonication.In some embodiments, method of the pores are induced by a pore-formingpeptide, an alkane, a detergent, and the like.

G-proteins can also be incorporated into the lipoparticles by using a Gprotein-Gag fusion protein. The fusion protein can contain an amino acidlinker to allow adequate mobility of the G protein component for itsinteraction with incorporated GPCRs. The Gag-G protein fusion proteincan be created by fusing a G protein to a Gag protein. In someembodiments, the fusion occurs at residue 1955 of MoMLV Gag.

In some embodiments G protein isotypes, such as Gα_(i), Gα_(s), Gα_(q),and Gα₁₂, are incorporated into lipoparticles.

G-protein can also be incorporated into lipoparticles by fusing them topartner GPCRs which are then incorporated into the lipoparticlemembrane. A G-protein isotype can be fused to a GPCR at the membraneprotein's C-terminus. The GPCR-G protein fusion protein is incorporatedinto lipoparticles using techniques to produce lipoparticles asdescribed herein and elsewhere.

G-proteins can also be incorporated into lipoparticles by fusing them toa non-GPCR transmembrane protein that can be incorporated into thelipoparticle, and that serves to anchor the G proteins to the innermembrane. A G-protein isotype can be fused to the C-terminus(cytoplasmic) of a single transmembrane protein (e.g. CD4).Alternatively, a fusion protein may contain a truncated version of CD4with a shorter C-terminus that can also be constructed and incorporated.The single transmembrane-G protein fusion protein can be incorporatedinto lipoparticles using techniques as described herein.

The present invention also provides methods for producing lipoparticlescomprising a G-protein and a membrane protein comprising: a) contactinga lipoparticle comprising a GPCR with a protein comprising a G-protein;and b) incubating said mixture under conditions resulting in productionof said lipoparticle. In some embodiments the G-protein is contacted bysonication, pore-forming peptidation (e.g. melittin), electroporation,transfection and the like. In some embodiments, the G-protein is anessentially pure G-protein.

In some embodiments, the present invention provides methods ofidentifying modulators of a GPCR comprising: a) contacting alipoparticle comprising a GPCR and a G-protein with a test compound; andb) measuring GPCR activity; wherein an increase in GPCR activityindicates that the test compound is an activator of GPCR activity or adecrease in GPCR activity indicates that the test compound is aninhibitor of GPCR activity. In some embodiments, the GPCR or G-proteinis a modified GPCR or a modified G-protein. In some embodiments, themodified GPCR is a fusion protein as described above. The lipoparticlescan also comprise a hydrolysable or non-hydrolysable analog that can befluorescently labeled. In some embodiments, a GTP analog is GTP.

By using a lipoparticle that comprises a GPCR, a G-protein, a GTPanalog, or combinations and subcombinations thereof, the GPCR signalingactivity can be measured by measuring changes in fluorescence that occurwhen proteins come in contact with one another. For example, asdescribed above, if the GPCR and/or the G-protein is a fusion proteincomprising a fluorescent protein, the changes in fluorescence indicatesa change in GPCR activity. If the GTP analog is fluorescently labeled,when it binds to the G-protein, a change in fluorescence will occur,which is indicative of GPCR signaling activity. The changes influorescence can be easily measured using standard techniques andequipments, such as a fluorometer. Dyes can also be used to measure GPCRactivity. The dyes can be, for example, pH sensitive or voltagesensitive. The lipoparticles can be suspended in a concentrated solutionof a fluorescent dye (e.g. di-4-ANEPPS), which diffuses preferentiallyinto the lipid bilayer of cell membranes and is incorporated into thelipoparticles. Examples of alternative voltage-sensitive fluorescentdyes include, but are not limited to, di-4-ANEPPS(C₂₈H₃₆N₂O₃S),di-8-ANEPPS, rhodamine 421, oxonol VI, JC-1, DiSC3(5), and the like(Molecular Probes, Inc.). The dyes can be measured ratiometrically,responding to increases in membrane potential with a decrease influorescence excited at approximately 440 nm and an increase influorescence excited at 530 nm. In some embodiments, the GPCR is CXCR4and the G-protein subunit is Gα_(z) However, any GPCRs, G proteins,membrane potential-responsive fluorescent probes, or pH-responsiveprobes can also be used. To test the function of the GPCR, thelipoparticles can be contacted or exposed to a GPCR agonist. Ligandbinding by the GPCR results in a change in the structural conformationof the receptor associated with G-protein dissociation. This results ina change in the electrical potential across the lipoparticle membrane,causing a detectable signal emission from the dye probe. In someembodiments, fluorescence emission is not altered in null-lipoparticles,or in lipoparticles treated with a GPCR antagonist. In some embodiments,fluorescence is measured in real-time, beginning prior to the additionof the agonist. In some embodiments, fluorescence is measured using aPerkin Elmer LS-50B fluorometer.

lipoparticles can also comprise a GPCR (i.e. CXCR4), a G-protein subunit(e.g. Gα_(z)), a GTP analog, an ion channel, or combinations thereof. Insome embodiments, the ion channel is an inwardly-rectifying potassiumchannels (GIRK), such as Kir3.x. A fluorescent dye (e.g. di-4-ANEPPS)can be loaded into this lipoparticle. In some embodiments, the dyefluoresces in a lipid environment and its fluorescence spectrum changesin response to fluctuations in membrane potential. The presence of theGPCR and the G-protein subunit CXCR4 and Gα_(z) in lipoparticles can beverified by Western blot using anti-G protein and anti-GPCR antibodies.One skilled in the art would recognize that alternative GPCRs, Gproteins, and membrane potential-responsive fluorescent probes couldalso be used. To test their function, the labeled lipoparticles areexposed to a GPCR agonist. In some embodiments, stimulation of the GPCRcauses dissociation of the G protein from GPCR and activation of the ionchannel. The resultant movement of ions causes an alteration to thelipoparticle membrane potential, leading to a change in the fluorescenceof the dye. In some embodiments, fluorescence emission is not altered innull-lipoparticles or in lipoparticles treated with a GPCR antagonist.Fluorescence can be measured in real-time before and after adding theagonist. Thus, lipoparticles can also be used to measure GPCR activationof ion channel activation.

Method of Generating the Lipoparticle of the Invention

The method of making a lipoparticle involves using a cell. Hence in someembodiments the method of making the lipoparticle involves expression ofat least a competent portion of the genome of an enveloped virus in acell.

As used herein, the term “competent portion” refers to the portion ofthe virus that is sufficient to cause the budding of lipoparticle from acell.

In some embodiments the lipoparticle comprises a membrane protein ofinterest. The membrane protein may be a normal component of the cell orit may be provided exogenously to the cell using, for example,transfection, viral infection, or other known molecular biologytechniques.

In some embodiments of the making of the lipoparticle, at least acompetent portion of the genome of a virus is provided to a producercell which comprises a membrane protein, and the producer cell isthereafter incubated under conditions which permit expression of thegene products encoded by the competent portion of the virus genome.These gene products include factors which facilitate the generation ofthe lipoparticle. In some embodiments, the gene products facilitate theassociation of the capsid-like particle with the cell membrane. In someembodiments, the virus is an adenovirus. In some embodiments, the virusis an alphavirus, such as semliki forest virus. In some embodiments, thevirus is a baculovirus. In some embodiments, the virus is a vacciniavirus or a herpes virus.

The producer cell need not normally comprise the desired membraneprotein of interest. Thus, in another example of making the lipoparticleof the invention, a producer cell is provided with at least a competentportion of the genome of a virus and a membrane protein of interest, andis thereafter incubated under conditions which permit formation of alipoparticle of the invention comprising a membrane protein. Thismethod, therefore, does not employ a producer cell which naturallycomprises the membrane protein of interest.

For lipoparticles that will be produced for long-periods of time, celllines can be established that both produce the competent portion of aviral genome and express high quantities of the membrane proteindesired. In some embodiments, to be converted to producer cells forlipoparticles, human primary cells can comprise the MLV structural genegag and/or the membrane protein of interest. Stable cells that expresseither the MLV structural gene gag or the membrane protein of interestcan be complemented with the other lipoparticle component. These genescan be delivered either by plasmid transfection, retroviral infection,adenoviral infection, or other common means of genetic transduction. Forexample, a stable cell line expressing a membrane protein of interestcan be infected with an adenoviral construct expressing MLV gag in orderto produce lipoparticles. Different existing gene delivery systems canbe combined to produce new, and possibly more powerful, gene deliveryconstructs.

The present invention also provides for the use of viral vectors such asadenovirus and semliki forest virus to produce MLV structural geneswithin cells that naturally express membrane proteins of interest, forexample, but not limited to primary cells, hybridomas, stem cells,treated cells, or cell lines.

Capture of naturally expressed membrane proteins can result inpopulations of membrane proteins that better represent native membraneprotein structure. For example, some proteins are modified aftertranslation, and this modification may depend on factors within thecell. For example, CCR5 is sulfated on Tyrosine 11 and this sulfation isknown to alter its structure and make it more competent for interactionwith HIV-1 Envelope proteins. Similarly, CXCR4 is glycosylated, and ifthis glycosylation is removed, additional structures of CXCR4 areexposed. Different cell types are known to have different conformationsof the CXCR4 membrane protein, although it is not clear why someconformations are more prevalent on some cell types. Other cell typescan be induced (e.g. with hormones, growth factors, cytokines, orchemicals) to differentiate or change, often resulting in a change inthe membrane proteins at the surface of the cell. Different cell typescan also contain different transcriptional splice variants of the samegene. For example, CCR2 has two cell-type specific splice variants,CCR2a and CCR2b, which have differences in their C-terminus.

Other cell-specific interactions can also alter the epitopes of membraneproteins. For example, the coupling of G-proteins to a receptor canchange the conformation of the membrane protein (GPCR) that theG-proteins bind. Similarly, binding of the membrane protein CD4 by theintracellular protein Lck can influence the location and function ofCD4. One skilled in the art would recognize that lipoparticles can begenerated from any cell type by over-expressing a retroviral Gag proteinand harvesting the lipoparticles that are generated from these cells.The MLV Gag protein can be introduced using either DNA plasmidtransfection or using Ad-Gag infection.

As used herein, the term “Ad-Gag” refers to an adenovirus that expressesthe Gag protein. In some embodiments, the Ad-Gag adenovirus may alsoexpress other proteins, such as a protein that is incorporated into thelipoparticle.

In some embodiments, Ad-gag is used to infect a cell type of interest,the adenovirus expresses the gag gene and lipoparticles are produced,and as the lipoparticles particles bud from the surface of the cell,they incorporate the membrane proteins naturally expressed orexogenously expressed on the surface of the cell. Adenoviral vectors arean attractive choice for the transfer of the MLV gag gene into targetcells since they have proven gene transfer efficiency and canaccommodate a large insert. Lipoparticles derived from primary cellsfunction as a stable source of membrane proteins from these cells,without the limitations of living cells, protein degradation, orcontaminant proteins. Lipoparticles are especially useful if the primarycells have a limited lifespan. Such lipoparticles can be used to makeMAbs against the membrane proteins, discover drugs against the membraneproteins, or to identify orphan receptors on the cells. Primary cellswith interesting characteristics, such as the ability to bind a ligandof interest, are of especial note in using the lipoparticles foridentifying orphan receptors or unidentified receptors.

The cell type is not a critical factor and may be any cell of interest.For example, the cell type used to produce lipoparticles in order tocapture their native membrane proteins can include cell lines, primarycells, non-transfectable cell types, or hybridomas. As used herein, theterm “non-transfectable cell type” refers to a cell type that is unableto efficiently take up exogenous DNA via chemical or liposomal mediatedtransfected (i.e. calcium phosphate mediated and Lipofectin™,transfection reagent, mediated transfection, and the like). In the caseof hybridomas, the membrane protein of interest can be a membrane-boundantibody that would be incorporated into the lipoparticle. In otherembodiments, the antibody can be a whole antibody or a fragment of anantibody such as a Fab fragment, an immunoglobulin-fusion protein, asingle-chain Fv, an Fc-fusion protein, or combinations thereof. Oneskilled in the art will also recognize that the membrane protein neednot be naturally expressed within the cell type at all periods of time.The cell type can be induced, chemically or genetically, or treated(e.g. differentiated) in order to express the desired membrane protein.For example, heat shock can induce expression of a novel set of membraneproteins on the surface of many cells. In other cases, DMSO, hormones,hypertonic shock, growth factors, or other means can induce a cell typeto express a different set of membrane proteins.

Moreover, the invention further includes a composition comprising alipoparticle comprising a protein that spans a membrane at least once,where the lipoparticle is attached to a sensor surface. Such proteinscan interact to form complexes or otherwise interact while present inthe lipoparticle lipid bilayer.

One skilled in the art would appreciate, based on the disclosureprovided herein, that the lipoparticle can comprise any membraneprotein, i.e., any protein that typically is associated with a membrane.In some embodiments, the lipoparticle comprises a multiple membranespanning protein. That is, the protein spans the membrane at leasttwice. Such multiple membrane spanning proteins encompass a wideplethora of membrane proteins including, but not limited to, the 7transmembrane receptor proteins (e.g., G-protein coupled receptorproteins, GPCRs, which include chemokine receptors), ion channels,transporters (such as amino acid transporter MCAT-1, and the like).However, the lipoparticles may also comprise proteins that are targetedto the membrane by virtue of a signal peptide, whereas the protein wouldnot normally be present at the membrane.

In other embodiments making a lipoparticle further comprises providingan additional component to the producer cell, whereby, upon formation ofthe lipoparticle, the lipoparticle comprises the additional component.The additional component may be any molecule which can be provided tothe cytoplasm or the membrane of the producer cell. By way of example,the additional component may be a nucleic acid, an antisense nucleicacid, a gene, a protein, a peptide, Vpr protein, an enzyme, anintracellular antagonist of HIV, a radionucleotide, a cytotoxiccompound, an antiviral agent, an imaging agent, or the like.

Inclusion of the additional component in the lipoparticle may beaccomplished by directly coupling the additional component to thecompetent portion of the genome of the virus. For instance, if thecompetent portion of the genome is provided to the producer cell in theform of a plasmid, the plasmid may comprise a gene encoding an imagingagent or a reporter molecule, such as luciferase or green fluorescenceprotein. Inclusion of the additional component in the lipoparticle ofthe invention may also be accomplished by directly coupling theadditional component to a nucleic acid encoding the membrane protein.For example, if the membrane protein is provided to the producer cell inthe form of a DNA molecule encoding the same, an additional componentcomprising a protein may be provided to the producer cell by includingthe sequence of a gene encoding the protein in the DNA molecule, priorto provision thereof to the producer cell.

The additional component may also be provided directly to the membraneor the cytoplasm of the producer cell by, for example, including theadditional component in the extracellular medium of the producer cell.

It will be appreciated by one skilled in the art that the genes chosento construct a lipoparticle, such as a retroviral gag gene and/or adesired membrane/cellular protein gene, can be delivered to a cell inany number of ways. In some embodiments, the method of nucleic acidtransduction involves DNA transfection, such as by calcium phosphateprecipitation, lipid-based transfection, electroporation,microinjection, or other methods of DNA delivery. In another embodiment,the genes of interest are stably incorporated into the cell of interestby transducing or transfecting the cell with the genes of interest andthen selecting cells that express desired levels of the genes ofinterest.

In some other embodiments, the method of gene transduction involvesinfection of a desired cell with a viral vector. The viral vector can becomposed of any number of common viral vector gene delivery systems,such as adenovirus, adeno-associated virus, herpes virus, retrovirus,vaccinia virus, or alphavirus. Some of the most versatile viral vectorsavailable are adenovirus vectors that are capable of infecting nearlyany cell type and expressing high levels of a desired gene. Adenovirushas previously been used by others to produce large amounts ofinfectious retroviral vectors by placing the retroviral structural genes(gag and pol) inside an adenoviral vector (Duisit et al., 1999; Lin,1998). However, this system has never been used to producenon-infectious retroviral particles comprising membrane proteins, andthe quantity of particles (not just infectious units) has never beenreported. Adenovirus-based production of lipoparticles allows convenientgene transduction into a wide variety of cell types (including non-humancells), but also enables more convenient cell growth within rollerbottles and/or continuous flow systems. Alternative viral transductionvectors are also available (e.g. vaccinia, bacculovirus, semliki forestvirus, adeno-associated virus).

In other embodiments, the vector is a chimeric viral vector. A chimericviral vector contains viral nucleic acid sequences from two differenttypes of viruses. In some embodiments, the chimeric viral genomeincludes nucleic acid sequences from a “primary” virus that allows fortransfection of a variety of cells, including, but not limited toprimary cells, stem cells, hybridomas, cell lines, and the like. Thechimeric viral genome also includes nucleic acid sequences from a“secondary” virus. In some embodiments these secondary virus sequencesis the retroviral (e.g. MLV) gag gene. In some embodiments the primaryvirus is adenovirus. In some embodiments, this viral vector comprises anadenovirus that expresses retroviral gag (Ad-gag). In some embodiments,the viral vector comprises an adenovirus that expresses both theretroviral gag and pol genes (Ad-gag/pol). In some embodiments, theviral vector comprises an adenovirus that expresses gag and the proteasefragment of the pol gene (Ad-gag/PR). In some embodiments, the chimericviral vector comprises nucleic acid sequences from an adenovirus, aretrovirus, and a nucleic acid molecule encoding a cellular protein. Insome embodiments, the chimeric viral vector comprises a sequenceencoding Gag, but doe not comprise a sequence encoding the envelope,promoter, or packaging signal of a retrovirus. In some embodiments, achimeric viral vector does not comprise a retroviral sequence thatencodes a functional pol gene.

The following can be performed to construct and package a chimeric virusfor infection of producer host cells and production of secondary virus.Methods for constructing a recombinant Adenovirus are well described inthe literature (Ausubel, et al. (2001), Current Protocols in MolecularBiology), and several kits are available to construct such a chimericvirus (Invitrogen, Clontech). In one embodiment, a chimeric viral genomeis prepared by first isolating the constituent nucleic acids. Thenucleic acids are then joined, for example, using restrictionendonuclease sites at the ends of the molecule. The recombinant moleculeis ligated into a suitable plasmid or vector. Methods for preparing arecombinant nucleic acid are known by those skilled in the art (seeSambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989),(Ausubel, et al. (2001), Current Protocols in Molecular Biology)). Inanother embodiment, chimeric viral genomes are constructed usingtransfer vectors and homologous recombination.

Any primary virus can be used as the source for the primary viral genomein the chimeric virus. In some embodiments, the primary virus has abroad host range. Primary viruses include, e.g., adenoviruses,adeno-associated virus, retrovirus, alpha virus, vaccina viruses, andherpes simplex viruses (HSV). In some embodiments, primary virus nucleicacid sequences include those derived from the family Adenoviridae (White& Fenner, Medical Virology (4th ed., 1994)). One embodiment of theprimary virus is a primary viral genome derived from the strainadenovirus 5 (for the sequence of adenovirus 5, see Chroboczek et al.,Virology 186: 280-285 (1992)).

Typically, the chimeric virus is replication deficient (it cannotproduce additional primary virus) and lacks a gene required forreplication or packaging. For example, adenoviral vectors usually havethe E1A gene deleted from their genome. This gene is essential for viralreplication and is complemented by the packaging cell line 293. Thus, areplication deficient chimeric viral genome is packaged afterintroduction of the viral genome into 293 cells. Other genes that arecommonly deleted in adenovirus vectors are E1B and E3.

In some embodiments, the adenoviral vector can be used to express boththe structural protein (i.e. gag) and the desired membrane/cellularprotein. In some other embodiments, two different adenoviruses are usedto express the structural protein and the desired membrane/cellularprotein. In some embodiments, cells can be infected with equivalentquantities of Adenovirus-Gag and Adenovirus-cellular protein, but theratios of the two viruses can be varied in order to express morecellular protein per lipoparticle (e.g. a cellular protein:Gag vectorratio of 3:1) or to produce more particles relative to receptor (e.g. acellular protein:Gag ratio of 1:3). In some embodiments the ratio ofAd-cellular protein to Ad-Gag is about 1:100, about 1:10, about 1:5,about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about4:1, about 5:1, about 10:1, or about 100:1.

In making the lipoparticle, the identity of the producer cell which isused is not critical. In some embodiments, the cell is an immortalizedcell line, such as HEK-293, HEK-293T, Cos, HeLa, CHO, 3T3, QT6, Cf2TH,CCC, Vero, or BHK. In some other embodiments, the cell is a non-human,human, stem, or hybridoma cell. The production of a human membraneprotein in a non-human cell type provides the opportunity to study thatmembrane protein isolated away from components that it might otherwisenaturally interact with. In some embodiments the human membrane proteinis incorporated into a lipoparticle produced from a mouse cell type. Insome embodiments the cell type is from the same species that is used forlipoparticle immunization. By using a cell type that is from the speciesthat is used for immunization, the immune response would be focused onthe membrane protein instead of membrane proteins from the producercell. Cell types that can be used for lipoparticles to use inimmunization can be any cell type, but in some embodiments the cell typeis derived from a Balb-c mouse.

In another embodiment, the cell is a primary cell type derived fromanimal tissue such as liver, spleen, brain, pancreas, intestine, skin,or bone. In another embodiment, the cell is a stem cell in which thecell is capable of differentiating into a number of type-specific cells.In another embodiment, the cell is a differentiable cell type in itspre-differentiated form or in its post-differentiated form. Theproduction of lipoparticles from a cell before and after differentiationwould provide the means to compare membrane proteins from these twocellular conditions. As used herein, the term “primary cell” refers to acell that has not been immortalized or is not immortalized (i.e. willundergo a finite number of cell divisions, usually less than 100, butcan be more.)

The production of lipoparticles from many cell types can be difficultwithout a high efficiency means of gene transduction. In some cases,plasmid transfection can achieve high efficiency gene transduction (>50%of cells). In other cases, plasmid transfection is inadequate or isprohibitively expensive to perform. For example, many primary cell typesare not readily transfected with calcium phosphate or other means ofplasmid transfection. In these cases, viral vectors can provide a meansto express the structural proteins of a lipoparticle (e.g. Gag) and/orthe membrane protein of interest within the cell type of interest. Manyviral vectors can be used, including adenovirus, adeno-associated virus,herpes virus, retrovirus, vaccinia virus, or alphavirus. In someembodiments, adenovirus is used to express the gag gene of a retrovirus.In some embodiments, a retroviral vector is used to express the gag geneof another retrovirus. In some embodiments, the retroviral vector is alentiviral vector that is capable of entering and expressing withinquiescent cells. The use of these viral vectors to express the genesused in the lipoparticle enables a number of new cell types to be usedin the production of lipoparticles.

In some embodiments, structural proteins from other virus families canbe used. Examples of other virus families include, but are not limitedto, alphavirus, arenavirus, arterivirus, bomavirus, bunyavirus,coronavirus, filovirus, flavivirus, hepadnavirus, herpesvirus,orthomyxovirus, paramyxovirus, poxvirus, retrovirus, rhabdovirus,togavirus, adenovirus, astrovirus, calcivirus, papillomavirus,parvovirus, picornaviridae, polyomavirus, reovirus. In some embodiments,structural proteins besides gag can be used. Examples of otherstructural proteins include, but is not limited to, nucleoprotein (NP),nucleocapsid, nucleoprotein (N), capsid protein, C protein, hepatitisdelta antigen), Core proteins (i.e., A3L, A4/5L, A10L, D2L, D3R, orF17/18R), and the like.

Depending on the structural protein that is used, the protein caninfluence where the lipoparticle will bud from. In some embodiments thelipoparticle will bud from the golgi apparatus, the endoplasmicreticulum intermediate compartment, the endoplasmic reticulum, theplasma membrane, or the rough endoplasmic reticulum.

Conditions that enable formation of lipoparticles are well known in theart. These conditions may vary depending upon the properties of theproducer cell and the virus used. A number of references exist whichdescribe conditions which are useful for culturing particular envelopedviruses (Fields Virology, 3rd ed., Fields et al., eds., Lippincott-RavenPublishers, Philadelphia, Pa.). Particular non-limiting examples areprovided herein of conditions which are useful to enable formation oflipoparticles.

Conditions which enable formation of lipoparticles include conditionsthat enable expression of the competent portion of the genome of thevirus, conditions under which a membrane protein is present in themembrane of the producer cell, and conditions which enable the formationof lipoparticles from the components of a producer cell which has beenprovided with the competent portion of the genome. Details regardingprocesses by which enveloped viral particles are formed followingprovision to a cell of a competent portion of the genome of an envelopedvirus have been described in the art, for instance by Wiley (1985, inVirology, Fields et al., ed., Raven Press, New York, 45-52).

Lipoparticles can be produced in at least two different ways. First,production of large amounts of particles for high-demand applications(e.g. high throughput screening), and second, production of smallamounts of particles for limited use applications (e.g. testing of newreceptors). By choosing the appropriate production components, discussedbelow, both production scales can be accommodated while balancingdiffering needs for speed, cost, and up-front labor.

Although 150 mm dishes or flasks are used in many viral productionprotocols for production of non-infectious virus, more efficient systemsexist. In particular, cell factories (Nunc™) with multi-tiered growthlayers can be used for large-scale production. Another alternative is touse roller bottles, which can achieve 5-10 times the surface area of alarge dish. 293 cells are notoriously difficult to grow (poor adhesion)and transfect (poor efficiency) in roller bottles, but alternativeproduction conditions (described below) can make roller bottles morefeasible. Finally, for stable cells that secrete proteins or viruses,continuous flow systems have proven highly efficient. Cells within acontinuous flow chamber cannot be transfected, but for some transductionmechanisms (e.g. adenovirus infection, stable cells) such cultureconditions may be ideal.

The method of production of lipoparticles can be greatly influenced bythe cell type and gene transduction methodology used. For example,calcium phosphate transfection is efficient in only some cell types,such as HEK-293, and usually requires growing cells in an adherentfashion (e.g. within plates or flasks). More efficient means of growingcells for transduction and lipoparticle production, such as spinnerflasks and bioreactors, require non-adherent cells and also requiretransduction mechanisms more amenable to such growth conditions. Inthese cases, viral vectors can provide a means to express the structuralproteins of a lipoparticle (e.g. Gag) and/or the membrane and/orcellular protein of interest within the cell type of interest under thegrowth conditions desired. The use of a viral vector for genetransduction enables any number of cellular growth vessels andconditions to be employed that will not interfere with transduction orlipoparticle production. Many of these conditions are more convenient oramenable to large-scale production. In one embodiment, adenovirus isused to transduce the genes of interest into cells grown within abioreactor. In another embodiment, the cells can be grown and transducedin a spinner-flask. In another embodiment, the cells can be grown andtransduced in plates, flasks, roller bottles, or cell factories. The useof viral vectors to express the genes used in the lipoparticle enables anumber of new methods of cell growth to be used in the production oflipoparticles. Virus-based production of lipoparticles would not onlyallow convenient gene transduction into a wide variety of cell types(including non-human cells), but also enable more convenient cell growthwithin roller bottles and/or continuous flow systems.

In some embodiments, the lipoparticle is produced according to themethods described in US 2002/0183247A1, the entire contents of which arehereby incorporated by reference.

Applications

In some embodiments, lipoparticles can be used for isolating membranereceptor drug targets. Structurally complex targets include G-proteincoupled receptors (GPCRs), ion channels, and molecular transporters thatare involved in cell-cell recognition, cell-adhesion, lipidinteractions, and protein-protein interactions. More importantly,lipoparticles can also be applied to targets that are difficult orimpossible to work with using traditional systems. For example, targetswill include proteins that are toxic to cells, difficult to express, arehighly charged, have intractable or no identified ligand, or that arelocated on intracellular membranes. Lipoparticles can address each ofthese limitations. The production of lipoparticles using viral vectorswill allow better lipoparticles to be produced, since difficult membraneproteins, even those residing on primary cells, can be incorporated intolipoparticles. The types of targets that can include the following, butare not limited to the following examples.

Successful targets such as GPCRs, ion channels, and transporters maycomprise a major target class. Lipoparticles may include members of thisclass that are toxic to cells, have weak or no ligands, or that aredifficult to express or work with.

Some targets that are located on the cell surface are relatively simpletransmembrane molecules, spanning the lipid bilayer only once, yet arefunctional only in a multimeric complex composed of two or moremolecules. This complex can either be a complex of one molecule withother identical molecules (a homocomplex) or with different molecules (aheterocomplex). Important molecules that fall into this class includereceptor kinases such as EGF, integrins, and most viral envelopeproteins.

Ninety-five percent of a cell's membrane structures are estimated to liewithin the cell, in organelles such as the mitochondria, nucleus, golgi,and endoplasmic reticulum (ER). These membranes are filled withregulatory receptors and transporters, but have traditionally beeninaccessible for drug screening. Lipoparticles will be able to be usedto isolate these intracellular integral membrane proteins on the surfaceof a lipoparticle, thereby making them amenable to drug screening,binding assays, and protein interaction mapping.

In some embodiments, lipoparticles can be used for the development oflead drug compounds to complex targets. Applications within the drugdiscovery pipeline include, without limitation, the following.

Mapping the network of which proteins bind to which receptors is acritical step in defining optimal targets of the proteome. Mappingproteomic interactions and ligand fishing can be an application forlipoparticles.

High throughput screening methods used today are fast, but are usuallylimited to biologically manageable targets such as soluble molecules(e.g. enzymes) and readily accessible cell-surface molecules. Proteinsthat are toxic to cells, are difficult to express, are highly charged,have intractable or no identified ligand, or that are located onintracellular membranes are not amenable to most high throughput screensavailable today. Lipoparticles can address each of these limitations.

Simple yes/no binding information is not sufficient for leadoptimization. Data-rich information such as selectivity, specificity,affinity, and kinetics of interaction are standard measurements used torefine a drug candidate, but are not easily obtained, especially fordifficult targets. The use of lipoparticles on biosensors and data-richdetection devices enables more information to be gathered during leadoptimization.

Lipoparticles permit specificity of interaction to be measured usinghighly sensitive detection devices and using lipoparticles that mimiccell surface molecules that are known to influence absorption,distribution, metabolism, elimination, and toxicology (ADMET)properties.

The lipoparticles can also be used to assess the binding of the membraneprotein presented in the lipid bilayer of the particle with a testcomponent, and/or to assess the effect of a test compound on the bindingof the protein with a cognate ligand. This is because, as more fully setforth elsewhere herein, the protein embedded in the lipoparticle retainsits ability to bind with its cognate ligand(s) and because the protein,now present in a lipoparticle, can be used in assays where solubleproteins or whole cells cannot be used, such as assays where the proteinof interest must be bound to a support or substrate, including, but notlimited to, an assay using a microfluidic device, e.g., a biosensorassay.

In some embodiments, a lipoparticles is attached to a surface and thenis contacted with a ligand and the biosensor detects the binding to themembrane protein in the lipoparticle. The detection can be by surfaceplasmon resonance, colorimetric diffraction grating, deflection ofmicrocantilevers (Wu, G., et al., Nature Biotechnology 19, 856-860;Weeks B L et al., Scanning. 2003 November-December; 25(6):297-9), oracoustic wave response (Cooper, M. A., et. al. (2001). NatureBiotechnology 19, 833-837). Or in some embodiments, the ligand isattached to a surface that is part of a sensor and this is contactedwith a lipoparticle and the binding is detected. The detection can be bysurface plasmon resonance, colorimetric diffraction grating, deflectionof microcantilevers, or acoustic wave response. In some embodiments, theligand and lipoparticle are contacted in solution.

Biosensor devices are designed to measure the interaction betweenbiological molecules. Typically, biosensors measure direct interactionsbetween a protein of interest and potential ligands (proteins,antibodies, peptides, small molecules) that may bind to it. Biosensorsare typically highly sensitive and can work with and detect even veryweak or very small quantity interactions. Biosensor devices have beenconstructed that consist of optical chips, fiber optics, spectrometerdetectors, microchannel chips, nanowells, and microcantilevers, acousticwave devices. In some embodiments, the assay comprises using a biosensordevice wherein the device is a surface plasmon resonance biosensordevice.

In some embodiments, the lipoparticle is attached to a sensor surface,where a “sensor surface” is any substrate where a change in a propertyof the substrate mediated by the contacting of the surface with amolecule or compound is detected and can be compared to the surface inthe absence of such contacting. However, in other embodiments, thelipoparticle is already attached to a sensor surface. While the sensorsurface can be a biosensor chip as exemplified herein, the sensorsurface is not limited to such a chip. A sensor surface also includesany biosensor chip that is disclosed herein (e.g., a BIACORE C1 chip, aF1 chip, and the like), known in the art, or to be developed in thefuture. Such sensor surfaces include, but are not limited to, a glasssubstrate comprising a coating of, e.g., gold, which can furthercomprise, for instance, a dextran matrix. However, the invention is notlimited to any particular sensor surface. The important feature of sucha surface is that a change in a characteristic of the sensor surfacee.g., its refractive index, can be detected, preferably by an instrumentconnected to the sensor surface, such that data or information from thesensor can be assessed thus detecting the change, or lack of change, ofthe characteristic of the surface.

However, the present invention is not limited to any particular assay.Rather, the present invention encompasses any assay where the protein ofinterest is a membrane component and where study of the binding of theprotein with a ligand requires, or is facilitated by, presenting theprotein in the context of a lipid bilayer and/or attaching the proteinto a support or solid substrate. Such assays include, but are notlimited to, assays using a microfluidic device, an optical biosensor,PATIR-FTIR spectroscopy, which is a type of biosensor using totalinternal reflection Fourier-transform infrared spectroscopy (1998, Chem.Phys. Lipids 96:69-80), CPRW Biosensor (Coupled plasmon-waveguideresonance (CPWR) spectroscopy as described in Salamon et al. (1997,Biophys J. 73:2791-2197) and Salamon et al. (1998, Biophys J.75:1874-1885), Multipole Coupling Spectroscopy (MCS) as described inSignature Biosciences, on the worldwide web at signaturebio.com, Fiberoptic biosensors (Illumina) as described in Walt (2000, Science287:451-452) and Dickinson et al. (1996, Nature 382:697-700), Michaels(1998, Analytical Chemistry 70:1242-1248), Lab-on-a-chip microfluidics(manufactured by, e.g., Caliper and Aclara) as described in Sundberg etal. (Current Opin. in Biotech. 11:47-53), and Bousse et al. (1999,Electrokinetic Microfluidic Systems, SPIE Microfluidic Devices andSystems II 3877:2-8, Sep. 20, 1999-Sep. 21, 1999), Microchannels (Gyros'microchannels etched into a Compact Disc-based device) as described onthe worldwide web at gyros.com, Microcantilevers (Protiveris) asdescribed in Tamayo et al., 2001, Ultramicroscopy. 86:167-173), Wu etal. (2001, Nature Biotechnol. 19:856-860), Confocal microscopy andnanowell detection as described in Hunt et al. (InternationalPublication No. WO 01/02551), and Microwell binding assays. Theaforementioned, as well as similar assays known in the art or to bedeveloped in the future, are encompassed in the invention.

In some embodiments, a sensor surface comprises a 96-well, 384-well,1536-well, a nano-well, optical fiber, or slide format. In someembodiments, the surface comprises gold, glass, plastic, or acombination thereof.

The nature of the instrument or the particular surface to which thelipoparticle is attached is not crucial. That is, while a derivatizedgold surface or a short carboxy detran matrix can be used to attach thelipoparticle thereto, the invention is in no way limited to thesesurfaces; instead, the invention includes any surface that can be usedin a microfluidic device to assess the interaction of proteins. Suchsubstrates include, but are not limited to, a plethora of biosensor“chips” that are commercially available, and others surfaces that areknown in the art, or such surfaces as will be developed in the future.

As discussed above, lipoparticles can be used for the presentation ofstructurally intact membrane proteins for antibody generation.Antibodies are now in use throughout the biotechnology industry astherapeutics, diagnostics, and research and development reagents, andare a part of vaccine elicitation.

The lipoparticle allows the stable presentation of structurally intactmembrane proteins within a particulate format that is suitable forantigen presentation. That is, because the structure of complex membraneproteins can be maintained using the lipoparticle, the present inventionprovides for methods of using lipoparticles comprising a membraneprotein of interest as an immunogenic vector for production ofantibodies that specifically bind with the membrane protein. In someembodiments, the antibodies produced by this method can bind with theprotein in its native structure and thus can provide a method forproducing antibodies that can, for instance, inhibit protein function bysteric blocking of important sites on the protein and/or antibodies thatcan affect protein function by allosteric effect. The production oflipoparticles using viral vectors will allow better lipoparticles to beproduced for antibody purposes, since additional cell types (e.g.non-human mouse cells) can be used for incorporation of human membraneproteins.

The particulate nature of lipoparticles makes them comparable tokilled-virus vaccines currently used to successfully elicit immuneresponses (e.g. humoral and cellular). The ability to place non-viralmolecules within such an immunogen allows lipoparticles to have directapplication to both preventative and therapeutic vaccines. In someembodiments, the use of lipoparticles as an immunogen will elicit acytotoxic T-cell response as well as a humoral (antibody) response, bothof which can be important components of research and for vaccinepurposes.

The lipoparticle has additional uses beyond the applications describedabove. Even if expressed at low levels within tissues or cell lines,lipoparticles can be used to purify and concentrate targets to enabledrug discovery without the use of the cDNA clone. The production oflipoparticles using viral vectors will allow lipoparticles to beproduced using cell types with naturally occurring sources of desiredmembrane proteins. Lipoparticles also represent a method for purifyingmembranes and membrane proteins from any cell type, without lysing thecell and without contamination from such membrane preparation techniques(e.g. intracellular membranes, inverted membranes). Lipoparticles arestable and present correctly oriented membrane proteins.

Existing array technology is largely focused on oligonucleotide or cDNAarrays. More recent arrays have included antibody and protein arrays,but are limited to proteins that can be chemically stabilized on asurface while maintaining their structural integrity, requirements thatexclude integral membrane proteins. Lipoparticles can be used to presentintegral membrane proteins on a surface while maintaining theirstructural integrity. With an estimated 3,000-6,000 integral membraneproteins in the entire human genome, it is possible that the entirerepertoire of cell-surface receptors can be spotted on a single array,thus mimicking the cell surface. Integration of such array technologywith biosensors would create a detection system that recreates the cellsurface in vitro. Such arrays would be used for mapping protein-proteininteractions and drug screening.

Determination of the structure of integral membrane proteins iscomplicated by the multiple hydrophobic membrane spanning domains ofthis class of proteins. Because lipoparticles can be constructed withpurified receptor, they offer the opportunity to decipher the structureof the receptors in their native state. An NMR-based approach hassubstantial promise for this application and could have a major impacton the ability to design drugs against integral membrane proteins. Inaddition, an NMR-based approach is also capable of determining thestructure of ligands bound to their cognate receptors.

The initial focus of the biotechnology industry was on the developmentof proteins that could be directly used for therapeutic intervention.Proteins such as insulin, growth hormone, and IL-2 are examples ofsoluble molecules that are now in therapeutic use. Nearly all proteinsin development today as therapeutics are soluble molecules. Yet manymolecules that could have biological efficacy are not released from thecell membrane. Other molecules function only as homo- or hetero-dimers,and all molecules exhibit a greater avidity (and therefore potency) whenpresented as part of a multimeric complex. Lipoparticles can addressthese issues to enable a new class of proteins to be used astherapeutics. Moreover, the surface chemistry of lipoparticles can bealtered to give proteins embedded within them a longer circulationhalf-life in vivo.

Lipoparticles may be used to deliver a composition to a target cell.This composition delivery method is particularly useful when it isdesired to deliver a composition specifically to a cell which comprisesa viral envelope protein on its surface. Specific examples of such atarget cell include, but are not limited to, a cell infected with anenveloped virus, such as HIV, SIV, RSV, or ecotropic MLV. A target cellmay also be a cell infected with another enveloped virus vector of theinvention or a cell which has fused with an enveloped virus vector otherthan the enveloped virus vector of the invention.

As set forth herein, the lipoparticle comprising a membrane protein ofinterest can be used in a wide variety of applications. In someembodiments, the lipoparticle can be used in assays relating to, forexample, but not limited to, drug screening, peptide screening, agonistversus antagonist discrimination, ADMET studies, structure-activityrelationships studies, vaccine development, food testing, chemicalsensing, light sensing, content release, monoclonal antibody production,fusion studies, phage display methods, ligand “fishing” oridentification, protein interaction mapping, various diagnostics, andproduction of artificial cells, among many others. Such uses would beunderstood by the skilled artisan to be encompassed in the inventionbased upon the disclosure provided herein.

The present invention also provides for kits that comprise alipoparticle comprising a membrane protein, and/or compositions of theinvention, an applicator, and instructional materials which describe useof the compound to perform the methods of the invention. Althoughexemplary kits are described below, the contents of other useful kitswill be apparent to the skilled artisan in light of the presentdisclosure. Each of these kits is included within the invention.

In some embodiments, the kits of the present invention described hereinweigh less than 50 pounds, 20 pounds, 10 pounds, 5 pounds, 3, pounds, 2pounds, or 1 pound.

In one aspect, the invention provides for kits for assessing the bindinginteraction of a membrane protein with a ligand. The kit comprises alipoparticle comprising a membrane protein, a ligand of the membraneprotein, and a substrate to which the lipoparticle can be attached. Thekit further comprises an applicator, which applicator can be used toattach the lipoparticle to the substrate and/or for applying the ligandsuch that the ligand is contacted with the lipoparticle comprising themembrane protein. Such an applicator includes, but is not limited to, apipette, an injection device, a dropper, and the like.

In some embodiments, the kit comprises a lipoparticle already attachedto a substrate with or without the ligand being bound to the membraneprotein. The substrate can then be examined using methods well known inthe art to detect any change in the substrate mediated by or associatedwith the ligand binding with its cognate membrane receptor present inthe lipoparticle.

In one aspect, the surface includes a wide variety of sensor surfaces,such as, but not limited to, a plethora of biosensor chips that areknown in the art or to be developed in the future.

The present invention also provides for kits for identifying a potentialligand of a membrane protein. The kit comprises a lipoparticlecomprising a membrane protein. In some embodiments, the kit comprises alipoparticle that is attached to a surface and can also comprise alipoparticle that is provided separately from the surface, which is alsoprovided in the kit. In other embodiments, the kit further comprises atest ligand, or a plurality of such ligands, such as, but not limitedto, a library of test ligands to be assessed for their ability tospecifically bind with the membrane protein present in the lipoparticle.

The present invention also encompasses a kit where the lipoparticle isprovided physically separated from a ligand and where the ligand isalready bound with the lipoparticle. Similarly, the present inventionencompasses a kit where the lipoparticle is provided physicallyseparated from the surface, as well as a kit where the lipoparticle isprovided attached to the surface. Further, the present invention alsoencompasses a kit with all possible permutations such that the ligandcan be bound with the lipoparticle which is, in turn, attached to thesurface, or each is provided separately, or any permutation thereof.

The present invention also provides for kits for identifying a compoundthat affects binding between a ligand and a protein (i.e., a receptor).The kit comprises a lipoparticle comprising a protein and a surface towhich the lipoparticle can be attached. In some embodiments, the kitcomprises a lipoparticle and a surface that are provided separately orwhere the lipoparticle is provided already attached to the surface.

The invention also provides for kits to produce lipoparticles. In someembodiments the kit comprises a producer cell, at least one viralvector, and/or producer cell media. The vector may comprise a viralvector that is amenable to cloning a membrane spanning protein ofinterest into the vector. In some embodiments, the kit comprises a viralvector that expresses Gag. In some embodiments, the kit comprises avirus that is ready to infect a cell, wherein the virus expresses aviral structural core protein such as, but not limited to Gag. In someembodiments, the virus is an adenovirus.

Viral expression systems provide a convenient, scalable, andreproducible method for lipoparticle production. By using such viralexpression systems to both produce Lipoparticles and express membraneproteins, high levels of membrane protein can be produced and capturedwithin the lipoparticle. In some embodiments, simultaneous infectionwith recombinant virus vectors expressing Gag and a membrane protein canbe performed, but in some embodiments, staggered infections can be used.For example, expression of CXCR4 prior to Gag expression may ensure thathigh levels of CXCR4 are incorporated into all lipoparticles (whichbegin to be produced only once Gag is introduced). Similarly,reinfection (expression boosts) may result in further improvements inmembrane protein production. In some embodiments, mixed expressionsystems can be employed for lipoparticle production (e.g. adenovirusexpressing Gag and SFV expressing CXCR4).

In addition to x-ray crystallization studies, a number of alternativestructural analyses have also been used with membrane proteins and maybe used with lipoparticle-derived membrane proteins, including cryo-EM(Henderson, et al. (1990), J Mol Biol, 213:899-929), projection maps(Schertler, et al. (1993), Nature, 362:770-2), lipidic cubic phase(Nollert, et al. (2002), Methods Enzymol, 343:183-99), 2Dcrystallization, solution NMR, solid-state NMR (Opella, et al. (2002),Biochem Cell Biol, 80:597-604), atomic force microscopy (Werten, et al.(2002), FEBS Lett, 529:65-72), and electron tomography (Werten, et al.(2002), FEBS Lett, 529:65-72, Zhu, et al. (2003), Proc Natl Acad SciUSA, 100:15812-7) (reviewed in (Torres, et al. (2003), Trends BiochemSci, 28:137-44)). In addition, a host of structural techniquesapplicable to membrane proteins in lipid bilayers are emerging (Caffrey(2000), Curr Opin Struct Biol, 10:486-97). Several of these techniques(e.g. cryo-EM and tomography) utilize membrane proteins embedded inlipid bilayers, and have already been applied to the low resolutionexamination of whole-retroviral membrane protein structures (Zhu, et al.(2003), Proc Natl Acad Sci USA, 100:15812-7). The structure of ligands,peptides, or proteins binding to membrane proteins within thelipoparticle can be determined, for example by solution or solid-stateNMR, X-ray crystallography, cryo-EM, projection maps, lipidic cubicphase, 2D crystallization, atomic force microscopy or electrontomography. The lipoparticle offers significant advantages for all ofthese structural studies.

In some embodiments, the structure of ligands bound to lipoparticles,ligands bound to membrane proteins, membrane proteins, and otherproteins can be determined by, for example, x-ray crystallography,cryo-EM, projection maps, lipidic cubic phase, 2D crystallization,solution Nuclear Magnetic Resonance, solid-state Nuclear MagneticResonance, atomic force microscopy, electron tomography and the like.

For example, membrane proteins within lipoparticles are structurallyintact and derived from a homogeneous source. Because of the mechanismof lipoparticle production, only proteins in their fully-processednative form on the plasma-membrane are isolated. In contrast, lysatesfrom cells will contain a membrane protein of interest in all stages ofsynthesis, folding, and processing. Many of these proteins will notretain their native structure and complicate structural analyses byintroducing significant heterogeneity.

Additionally, the lipoparticle facilitates separation of membraneproteins from contaminating cellular structures. lipoparticles areharvested in culture supernatant, and can be readily separated fromdebris using simple centrifugation or chromatography steps. The use oflipoparticles as a starting material provides a higher quality proteinsource for subsequent purification steps. Purification techniques andreagents that might not be successful when starting with whole cellsmight succeed with lipoparticles.

Also, membrane protein can be produced from cells indefinitely.Traditional membrane protein expression systems using cells must beterminally harvested. Lipoparticle production itself is not toxic tocells, so lipoparticles can be harvested multiple times over longperiods of time. In effect, the lipoparticle mimics the production ofsecreted protein, providing a greater surface area of plasma membranefrom which to capture membrane proteins as new plasma membrane isgenerated and released with lipoparticles.

Methods and Devices for Detecting Proteins

In a 2000 paper published in PNAS, it was demonstrated that a number ofcomplex proteins, including GPCRs, can be incorporated intolipoparticles and attached to the BIACORE biosensor (Hoffman, et al.(2000), Proc. Natl. Acad. Sci. USA, 97:11215-11220). Binding ofantibodies, peptides, and proteins to these receptors exhibitedappropriate specificity, and structural integrity of the receptors wasmaintained. An affinity of interaction between a protein and itsreceptor (HIV gp120 and CXCR4) of approximately 500 nM was alsomeasured. This affinity is sufficiently weak so as to render previousstandard binding assays unable to detect the interaction (Doranz, et al.(1999), J. Virol., 73:10346-10358, Doranz, et al. (1999), J. Virol.,73:2752-2761). The affinity with which different HIV-1 gp120 proteinsbind to chemokine receptors can be an important determinant of viralpathogenesis, so measuring these interactions has enabled practitionersto begin correlating binding affinity with disease progression. The useof the lipoparticle made this possible, and provides a clear examplewhere use of this novel approach has proven its worth. Work with theGPCRs CCR5 and CXCR4 has, to date, been directly applicable to othermembrane proteins. Over twenty different membrane proteins have beenincorporated into virus-based lipoparticles, including GPCRs, ionchannels, transporters, and Type I and Type II single transmembraneproteins.

In some embodiments, the present invention relates to a compositioncomprising an array of lipoparticles attached to a surface or a sensorsurface. In some embodiments, the present invention relates to acomposition comprising an array of viral particles attached to a surfaceor a sensor surface. In some embodiments, the present invention relatesto an array of pseudotypes attached to a sensor surface. In someembodiments, the present invention relates to a composition comprisingan array of at least 1 lipoparticle and at least 1 pseudotype. In someembodiments, the present invention relates to a composition comprisingat least 1 lipoparticle, at least 1 pseudotype, at least 1 viralparticle, or combinations thereof.

As used herein, the term “array” refers to a group of at least 2members. In some embodiments, an array comprises at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 15, at least 50, at least 95, at least 96, atleast 384, at least 1,000, at least 5,000, or at least 10,000 members.As used herein, the term “member” refers to a lipoparticle, virus,pseudotype, as well as empty positions.

As used herein the term “positions” refers to distinct locations on asensor surface to which a lipoparticle, pseudotype, or viral particlemay be attached.

As used herein, the term “array of lipoparticles” refers to a group oflipoparticles.

In some embodiments, an array of lipoparticles comprises least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 95, at least 96, at least 100, at least 200, at least 300, atleast 384, at least 500, at least 1,000, at least 2,000, at least 5,000,or at least 10,000 positions.

In some embodiments, the array of lipoparticles comprises at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 95, at least 96, at least 100, at least 200, at least 300, atleast 384, at least 500, at least 1,000, at least 2,000, at least 5,000,or at least 10,000 lipoparticles.

In some embodiments, the group of lipoparticles comprises at least twolipoparticles that have different compositions. Therefore, in someembodiments, an array of lipoparticles refers to a heterogeneous groupof lipoparticles rather than a homogenous (i.e. a group of lipoparticleswhere all the lipoparticles contain the exact same membrane protein orexogenously expressed protein).

In some embodiments, an array of lipoparticles comprises least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 95, at least 96, at least 100, at least 200, at least 300, atleast 384, at least 500, at least 1,000, at least 2,000, at least 5,000,or at least 10,000 different lipoparticles. In some embodiments, anarray of lipoparticles comprises 95 or 96 different lipoparticles.

In some embodiments, the array of lipoparticles is arranged such thatthe spacing of the lipoparticles occurs along a horizontal axis and avertical axis, wherein each axis comprises at least 2 positions, suchthat the array is 2×2. In some embodiments, the array is in thearrangement of 8×12 (e.g. 96 well plate), 16×24 (e.g. 384 well plate),or 32×48 (e.g. 1536 well plate).

In some embodiments, an array of lipoparticles comprises 96 positions.In some embodiments, an array of lipoparticles comprises 96lipoparticles attached to a sensor surface. In some embodiments, anarray of lipoparticles comprises 95 lipoparticles attached to a sensorsurface and one empty position on the sensor.

In some embodiments, an array of lipoparticles attached to a sensorsurface comprises at least one position on the sensor surface that isleft empty. In some embodiments, at least 2, at least 3, at least 4, atleast 5, at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 91, atleast 92, at least 93, at least 94, or at least 95 positions are leftempty.

As used herein the term “an array of lipoparticles attached to a sensorsurface” refers to a group of lipoparticles that is attached to a sensorsurface and can include empty positions. In some embodiments thelipoparticles can be attached to the sensor surface by covalent bonds,ionic bonds, hydrophobic interactions, affinity interactions,hydrophilic interactions, chemical crosslinking, and the like. The exactmethod used to attach the lipoparticles to the sensor surface is notessential to the invention, as long as the integrity of thelipoparticles is, maintained.

The present invention also provides method of identifying a bindingpartner of a membrane protein comprising contacting a surface coatedwith a lipoparticle comprising the membrane protein with an arraycomprising the binding partner. In some embodiments, the surface mayalso be coated with a virus. By coating a surface with the samelipoparticle an array of other molecules can be screened to determine ifa member of the array can bind to the lipoparticle. Thus, while thelipoparticle composition is uniform the members of the array are not andthus, a large number of molecules can be screened against the surfacethat is coated with the lipoparticles. Methods of coating a molecule ona surface can routinely be done (see, for example, Gosalia D N, DiamondS L. Proc Natl Acad Sci USA. (2003) Jul. 22; 100(15):8721-6.)

As used herein, the term “sensor surface” is any substrate where achange in a property of the substrate mediated by the contacting of thesurface with a molecule or compound is detected and can be compared tothe surface in the absence of such contacting. In some embodiments, alipoparticle can be attached to a surface and then an interactionbetween a lipoparticle and a substrate is detected by another mechanism.In some embodiments, the substrate is attached to a surface.

While the sensor surface can be in the form of a chip as exemplifiedherein, the sensor surface is not limited to such a chip. Instead, theskilled artisan would appreciate, based on the disclosure providedherein, that a sensor surface includes not only any biosensor chip thatis disclosed herein (e.g., a BIACORE C1 chip, a F 1 chip, and the like),but also any biosensor chip presently known in the art, or to bedeveloped in the future. Such sensor surfaces include, but are notlimited to, a glass substrate comprising a coating of, e.g., gold, whichcan further comprise, for instance, a dextran matrix. The presentinvention is not limited to any particular sensor surface. The importantfeature of such a surface is that a change in a characteristic of thesensor surface e.g., its refractive index, can be detected, preferablyby an instrument connected to the sensor surface, such that data orinformation from the sensor can be assessed thus detecting the change,or lack of change, of the characteristic of the surface.

Example of biosensors and detection systems include, but are not limitedto, biosensors and systems created by Biacore™, Protiveris™, Luna™,Illumina™, SRU Biosystems™, Akubio™, Applied Biosystems™, Graffinity™,and HTS Biosystems™.

One skilled in the art will recognize that the optical biosensor is buta detection mechanism. Optical biosensor technology is a growing fieldwith increasing sophistication, and the present invention can bepracticed using a wide variety of biosensors. The present invention canbe practiced using any biosensor, including biosensors based on arrayingthe samples to an alternative surface (such as a glass slide), orbiosensors which have been reduced in size.

In some embodiments, the sensor surface comprises a sensor, a biosensor,an optical fiber, or a microfluidic device. The mechanism by which anoptical biosensor measures the interaction is also not essential to theinvention and can include any method. In some embodiments, an opticalbiosensor measures an interaction by surface plasmon resonance (SPR),colorimetric diffraction grating, chemiluminescence, fluorescence, andthe like.

In some embodiments the sensor surface can also comprise a 96-well,384-well, 1536-well, a nano-well, optical fiber, or slide format. Insome embodiments, the sensor surface comprises gold, glass, plastic, ora combination thereof. However, the exact materials that comprise thesensor surface is not essential as long as the sensor surface enablesthe detection or an interaction, and that such information can beassessed thus detecting the interaction, or change, the lack ofinteraction, or change.

The membrane proteins can be either cloned or obtained from commercialor academic sources and produced in lipoparticles as described herein.The structural integrity of membrane proteins within each batch oflipoparticles can be tested using conformationally-sensitive MAbs and/orligands, depending on availability.

The present invention also provides methods for detecting the structuralintegrity of a membrane protein within a particle (e.g. virus particleor lipoparticle) comprising contacting the particle with a molecule thatbinds to the membrane protein; and detecting binding of the molecule tothe particle, wherein the binding of the molecule to the particle isindicative that the structural integrity of the membrane protein isintact. In some embodiments, the molecule is an antibody,conformation-dependent antibody, ligand (e.g. toxin), agonist, orantagonist. In some embodiments, the method comprises a centrifugationstep. In some embodiments, the method of detection comprises aVirus-Detection ELISA, an antibody detection Viral ELISA, a sensor,flow-cytometry, immunofluorescence staining, centrifugation, orcombinations thereof.

In some embodiments, a lipoparticle has only one exogenous membraneprotein. Some lipoparticles, can, however, contain more than oneexogenous membrane protein. In some embodiments, the exogenous membraneprotein can be any membrane protein. In some embodiments, the exogenousmembrane protein is both alpha and beta dystroglycan, and the like.

The lipoparticles can be incorporated into the BIND™ biosensor detectionsystem from SRU Biosystems (Woburn, Mass.). BIND is a label-freebiosensor with continuous biosensor gratings that is compatible withindustry-standard 96-well and 384-well plates and microarray slides.SRU's technology is based on a colorimetric diffractive grating surfacethat, when illuminated with white light, is designed to reflect only asingle wavelength. When molecules are attached to the surface, thereflected wavelength (color) is shifted due to the change of the opticalpath of light that is coupled into the grating. SRU's biosensors havepicomolar sensitivity, and are not based on detection of mass, so theycan detect binding of low molecular weight compounds as easily as largeproteins. The chemical surface of the biosensor can be derivatized foroptimal attachment using much of the same chemistry as BIACORE chips. Asingle spectrometer reading can be performed in several milliseconds,allowing reaction kinetics to be monitored in real time.

In some embodiments, biosensors require that the protein of interest beimmobilized on the biosensor surface. One skilled in the art willrecognize that attachment conditions will vary depending on thebiosensor used.

The SRU biosensor uses a polymer surface that is modified to containcarboxyl groups for surface attachment. Aside from the biosensorsurface, the other major surface involved is on the lipoparticles.

The lipoparticles surface is similar to that of a cell, containinglipids, proteins, and carbohydrates, any of which can be readily used ormodified to facilitate attachment.

In some embodiments, the screening of samples on a SRU biosensor canfollow the following sequence: pre-blocking, blocking,application/binding of sample, and regeneration. For example, thebiosensor can be created to include lipoparticles containing anthraxtoxin receptors. In some embodiments, the biosensor can be preparedduring the pre-blocking stage with PBS for 10 minutes. Blocking can beperformed using 10% Serum in PBS for 5 minutes. Samples can be diluted1:1 with PBS and can be applied to the biosensor at a rate of 5 ul/minfor 10 minutes at pH 7.2. Regeneration of the biosensor can proceed withthree 30 second applications of Sodium Carbonate at pH 9 with 0.5 molarNaCl. The sequence can then be repeated for the examination of anothersample.

In some embodiments the array of lipoparticles comprises a lipoparticlethat does not contain a protein of interest. The lipoparticle that doesnot comprise a protein of interest can be referred to as a controllipoparticle. The lipoparticle that does not comprise a protein ofinterest can also be referred to as a negative control lipoparticle. Insome embodiments, the array of lipoparticles does not comprise a controllipoparticle. In some embodiments, the array of lipoparticles attachedto a sensor surface comprises a position on the sensor surface that is“empty.” As used herein, the term “empty” refers to a position on thesensor surface that does not contain a lipoparticle. In some embodimentsthe array of lipoparticles attached to a sensor surface comprises atleast one location that contains a lipoparticle with a specific membraneprotein. The specific membrane protein can be any membrane protein.

For example, the array of lipoparticles attached to a sensor surfacecomprises CCR5, CXCR4, DC-SIGN, DC-SIGNR, CFTR, CD44, mannose receptorMRC1, alpha dystroglycan, beta dystroglycan, the anthrax toxin receptor,and the like. In some embodiments, a single lipoparticle in the array oflipoparticles comprises one membrane protein, two, three, four, five,six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, onehundred, or more than one hundred membrane proteins. The lipoparticlescan comprise any membrane protein or protein that can be inserted into alipoparticle.

The lipoparticles can also comprise proteins derived only from naturallyoccurring sources. The lipoparticles can be produced from a primarycell, an organ, a stem cell, a cell line, and the like. With suchlipoparticles, the proteins are not exogenously added (i.e. they arenormally produced by the source.) The organ can be any organ from anorganism and includes, but not limited to, liver, kidney, brain,pancreas, intestine, skin, testes, ovaries, heart, lung, and the like.

In some embodiments, the lipoparticles are attached to the biosensorusing one of the following three methods: Amine coupling: (Parameters ofpH (5.5), ionic strength, coupling reagents (0.25 M EDC, 1.0 M NHS), andamine quenching (1 M ethanolamine, pH 8.5)); Avidin-Biotin attachment:Streptavidin can be amine coupled to the biosensor surface, andlipoparticles will be biotinylated by incorporation ofphosphatidylcholine-biotin lipids or alternatively by chemical couplingof biotin moieties; or Lectin binding: A wheat germ agglutinin (WGA)surface will be created by covalently coupling WGA (Vector Laboratories)to the biosensor surface. WGA is a lectin that binds glycolipids on cellmembranes and is routinely used for binding of membrane vesicles, andcan bind lipoparticles directly.

In some embodiments, lipoparticles, viruses, or virus-like particles canbe captured in similar ways. lipoparticles, viruses, or virus-likeparticles can also be captured to surfaces by mixing the particles insolution with a capture agent, such as WGA-biotin, and then flowing theparticles over a suitable surface, such as avidin. Lipoparticles canalso be captured using a membrane protein in the lipoparticle, such as atransmembrane-anchored avidin fusion protein or a fusion proteincontaining a His-tag, that allows attachment of the lipoparticle to asuitable surface such as biotin or Ni+2.

In some embodiments, the lipoparticles are attached as follows: Activatethe surface carboxyl groups with a 1:1 mixture of 1M1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) andNHS for 8 minutes. Lower the flow rate to 1 μL/min and inject thelipoparticle solution mixed 1:1 with 0.1 M sodium acetate, pH 5.5).Inject manually for about 20 minutes until the desired level ofattachment is achieved. Block with 1 M ethanolamine, pH 8.5 for 8minutes.

Lipoparticles can also be attached to the Biacore chip. Attachment oflipoparticles using microfluidic flow-cell delivery has already beenused within the BIACORE biosensor with success (Hoffman, et al. (2000),Proc. Natl. Acad. Sci. USA, 97:11215-11220). The microfluidic mechanisminherently keeps the sample uniform as it is delivered to the biosensorsurface.

To attach lipoparticles to a BIACORE chip the following procedure can befollowed. The BIACORE sensor surface uses a gold surface that iscovalently modified with free carboxyl groups, and we have successfullyused this surface (C1 and F1 BIACORE chips) for covalent attachment oflipoparticles via amine coupling. For amine coupling, the C1 biosensorchip surface can be activated with a 1:1 mixture of 0.25 M EDC and 1 MNHS. Lipoparticles that have been mixed 1:1 with 0.1 M NaOAc pH 5.5 canbe injected for 15 min at 1 μl/min. Following attachment, the remainingsurface carboxyl groups can be quenched with 1 M ethanolamine, pH 8.5.

In some embodiments, binding of conformation-dependent MAbs (Monoclonalantibodies) are used to ascertain successful attachment and retention ofmembrane protein conformation. A range of lipoparticles concentrations(0.1-10 μg) can be captured and compared. Lipoparticles without anymembrane protein can serve as a standard negative control.

The sensor surface can also be modified to bind lipoparticles. In someembodiments the sensor surface is modified with a composition comprisingpoly-lysine, alkane modification (the covalent attachment of hydrophobiccarbon chains), gamma-aminopropylsilane, histidine, protein A, lectin,avidin, streptavidin, acylation, Ni⁺⁺, and the like. The sensor surfacecan also be modified by any combination of compositions that willfacilitate the binding of lipoparticles. In some embodiments the lectinis wheat germ agglutinin. In some embodiments the sensor surface ismodified to preserve membrane protein structure. To preserve membraneprotein structure the sensor surface can be modified with, but notlimited to, trehalose, glycerol, sucrose, gelatin,gamma-amino-propylsilane, or a combination thereof.

As used herein, the term “alkane modification” refers to the covalentattachment of hydrophobic carbon chains.

In some embodiments, the lipoparticles are modified to facilitate theirbinding to the sensor surface. For example, if the sensor surface iscoated with avidin the lipoparticles can be modified to be biotinylatedso that the biotinylated bioparticles will bind to the avidin on thesensor surface. Alternatively, the lipoparticles can be coated withWGA-biotin which in turn can bind avidin or streptavidin surfaces. Thelipoparticles can be modified with other agents (e.g. Ni⁺⁺) as well solong as the integrity of the lipoparticle remains intact.

Once coated onto a surface lipoparticles containing a membrane proteincan be used, for example, to screen an array of antibodies. In someembodiments, the antibodies are allowed to bind to the lipoparticles andare then washed away. The binding of the antibodies can be detected bycoating the entire slide with a fluorescent secondary antibody thatrecognizes the first antibody. The slide is washed and spots that havebound antibody are detected by visualizing fluorescent spots. Methods ofvisualizing secondary antibodies are known to one of ordinary skill inthe art.

In some embodiments, a library may be composed of antibodies, hybridomasupernatants, drug candidates, or peptides. In some embodiments, thelibrary arrayed onto the Lipoparticles may also contain glycerol toprevent drying of the spots. In some embodiments, lipoparticles can becoated by covering the entire slide with a solution of lipoparticles insucrose, allowing lipoparticles to attach for 1 hour, and then removingthe lipoparticle solution. In some embodiments, the solution of sucrosecomprises about 0.1%, about 0.2%, about 0.5%, about 1.0%, about 2.0%,about 3.0%, or about 5.0% sucrose. In some embodiments, the solution ofsucrose comprises more than 0.1%, more than 0.5%, more than 1%, morethan 2%, more than 3%, more than 4%, or more than 5% sucrose.

One skilled in the art will recognize that lipoparticles modifiedchemically (e.g. PEGylation, biotinylation, fluorescent labels) andgenetically (elimination of retroviral protease to increase particleproduction) may be used in the creation of the biosensor. Furthermore,one skilled in the art will recognize that different methods forproducing lipoparticles than herein disclosed may be employed for thecreation of the bioparticles.

Although the array of lipoparticles can comprise proteins from onespecies, in some embodiments, the lipoparticles present in the array cancomprise membrane proteins from more than one species. For example, anarray of lipoparticles may contain 10 different lipoparticles, 5lipoparticles may comprise a human protein, while the other 5lipoparticles may comprise mouse proteins. The number of speciesrepresented by a lipoparticle is not limited. An array may compriselipoparticles that each have proteins from a different species. Thus insome embodiments, an array of lipoparticles may comprises lipoparticlescomprising proteins from one, two, three, four, five, six, seven, eight,nine, ten, or more than ten species.

In some embodiments, the array of lipoparticles comprises lipoparticlescomprising proteins that are homologues from at least two species.

The present invention can be used for many applications. Anon-exhaustive list of applications are described for example in Table2. There are other numerous applications for which the present inventioncan be used for that are not disclosed herein and are readily apparentto those of ordinary skill in the art and are included in the presentinvention.

TABLE 2 Specific examples of applications for the Receptor BiosensorClass Application Drug Discovery Refinement of lead compound specificity(lead optimization) Drug Discovery Identification of ligands to orphanreceptors (ligand fishing) Drug Discovery Identification of receptorsfor orphan ligands (de-orphaning) Drug Discovery Identification ofundesired binding reactions (toxicity correlates) Biomedical ResearchProteomic network mapping Biomedical Research Basis for in silicomodeling of biologically complex binding scenarios Diagnostics Dynamicmonitoring of immune response during disease Diagnostics Detection ofauto-antigen immune responses Diagnostics Detection of narcotics,pathogens, and biological warfare agents

Another aspect of the present invention relates to methods of detectinga ligand binding to a lipoparticle. In some embodiments, the methodcomprises the steps of contacting a ligand with an array oflipoparticles attached to a sensor surface and detecting the binding ofthe ligand with the array of lipoparticles. In some embodiments, themethod comprises the steps of contacting a ligand with an array of,lipoparticles, viruses, or virus-like particles attached to a sensorsurface and detecting the binding of the ligand with the array oflipoparticles, viruses, or virus-like-particles. In some embodiments theligand comprises a fluorescent tag, enzymatic tag, biotinylated tag, aradioactive tag, and the like. In some embodiments the method ofdetecting a ligand binding to a lipoparticle can be used to determinethe affinity of a ligand to a membrane protein present in alipoparticle. For example, an array of lipoparticles can compriselipoparticles containing the membrane protein of interest as well as atleast one lipoparticle without the membrane protein of interest or alocation on the array that is empty. The ligand is contacted with thearray of lipoparticles under conditions which enable the ligand to bindto the lipoparticle. Once the ligand binds to the lipoparticle thesensor surface measures the interaction from which the affinity of theligand is determined. The interaction can also be used to determine theon-rate, the off-rate or the specificity of the ligand. For example, todetermine the specificity of the ligand, the array of lipoparticles cancomprise at least one lipoparticle comprising a membrane protein and alipoparticle that does not comprises a membrane protein. In someembodiments, the array of lipoparticles can comprises at least onelipoparticle with a first membrane protein and at least one lipoparticlewith a second membrane protein. In some embodiments, the array oflipoparticles comprises about 5, about 10, about 20, about 30, about 50,about 100 lipoparticles comprising different membrane proteins. It isnot required that each lipoparticle comprise a different membraneprotein.

In some embodiments, the present invention provides methods of detectingligand binding partners comprising the steps of contacting a ligand withlipoparticles with test binding partners; and detecting binding of testbinding partners with said lipoparticles. In some embodiments, the testbinding partner comprises a fluorescent tag, enzymatic tag, biotinylatedtag, paramagnetic tag, a radioactive tag, or a combination thereof. Insome embodiments, the binding of the testing binding partner to thelipoparticle comprises scintillation proximity or filtration binding.

In some embodiments, determining the specificity of a ligand comprisescomparing the binding of a ligand to a location on the array thatcontains a lipoparticle with a specific membrane protein to the bindingof the ligand to a location on the array that contains a lipoparticlewithout any specific membrane protein (i.e. a different membraneprotein) or to a location on the array that does not contain alipoparticle.

When screening samples, ligands, and the like on a biosensor one ofskill in the art can follow the following sequence: pre-blocking,blocking, application/binding of sample, and regeneration.

One skilled in the art will recognize that to optimize the system, anumber of experimental conditions can be altered. Examples of conditionsthat can be altered include, but are not limited to, those disclosed inTable 3.

TABLE 3 Usage Conditions & Alternative Usage Conditions AlternativesPre-blocking None, BSA, Gelatin, Serum, Glucose Blocking agent in bufferNone, BSA, Gelatin, Serum, Glucose NaCl (mM) 0, 50, 150 Blocking time(min) 0, 5, 10, 30 Attachment flow rate (μl/min) 2, 5, 10, 30 CouplingpH 4, 5, 6, 7 Running buffer PBS, HBS, DMEM Dispersive agents None,Pluronics, Gum arabic Neutralization of charged lipids None, Polybrene,DEAE-Dextran Experimental flow rate (μl/min) 15, 30, 60 Particleconcentration (mg/ml) 0.05, 0.1, 0.2, 0.5, 1.0 Regeneration conditionslow pH, high pH, chaotropic agents, salt Control surface nolipoparticles, no receptor, irrelevant receptor

A biosensor using lipoparticles to detect interactions can also begenerated using a BIACORE system. In some embodiments, conditions forusing a BIACORE system are described, but not limited to, in Table 4.

TABLE 4 Conditions used with BiaCore System. Variable Current StatusCoupling reagent 0.25M EDC, 1M NHS, fresh preparation Chip type C1 >F1 > L1 > B1 Coupling rates Fast activation rate (10 μl/min), slowcoupling rate (1 μl/min), fast quenching rate (10 μl/min) resulted in 2×rate of attachment and 3× attachment level Lipoparticles High purity andhigh receptor density are better. Concentration has small effectSpecificity Demonstration of specificity using controls includingirrelevant MAbs, Lipoparticles with irrelevant membrane proteins, blankflow cells Buffer HEPES ≧ Tris > Phosphate Ionic strength NaCl 50-150mM, pH 6.0-7.0 Additives Stable to glycerol, sucrose, ethanol, DMSO, PEGBlocking reagents 1 mg/ml BSA Sensitivity Detection limit as low as 20pM (3.2 ng/ml) with MAb Orientation a) Lipoparticles can be captured onthe biosensor surface and MAbs flowed across (better quantization), andb) Lipoparticles can be flowed across a MAb surface (increasedsensitivity) Regeneration Na₂CO₃, pH 9 + 0.5M NaCl, supports up to 200cycles, baseline stability can be improved Ligand size 8 kDa naturalligand (SDF-1) can be detected

One skilled in the art will recognize that these conditions can bemodified to test for infectious agents, food-borne illnesses,water-borne illnesses and contaminants, and other disease causing agentsnot described herein, and are within the scope of the present invention.The array of lipoparticles attached to a biosensor can also be modifiedto incorporate any membrane protein of interest or protein that isassociated with the membrane. Example of Water-borne illnesses andcontaminants are listed in Table 5

TABLE 5 Water Contaminants Type of Water Contaminants: Examples:Coliform bacteria Fecal Coliform and E coli Turbidity CryptosporidiumGairdia lamblia Inorganic Contaminants Arsenic Fluoride Lead SyntheticOrganic Contaminants, including Dioxin pesticides & herbicides EndothallPCBs Volatile Organic Contaminants Carbon Tetrachloride TolueneDisinfectants Chlorine Chloramine Disinfecting Byproducts Chlorite MTBE

The present invention also relates to methods of detecting an infectiouspathogen. In some embodiments a method of detecting an infectiouspathogen comprises the steps of: a) contacting a sample with an array oflipoparticles attached to a sensor surface, wherein the array oflipoparticles comprises membrane proteins that interact with infectiouspathogens; and b) detecting an interaction with said array oflipoparticles, wherein the detection of an interaction indicates thepresence of an infectious pathogen.

In some embodiments, the creation of a biosensor to detect aninteraction between a lipoparticle and a binding partner comprise: 1)selection of membrane proteins; 2) production of lipoparticlescontaining membrane proteins; 3) selection of biosensor surface and/orsystem; 4) attachment of lipoparticles to the biosensor surface; and 5)screening of samples.

In some embodiments, a sample can be contacted with an array oflipoparticles. The lipoparticles can comprise membrane proteins thatinteract with agents. These include receptors that interact withinfectious pathogen proteins, toxins, and the like. The infectiouspathogen can be any pathogen, including, but not limited to bacteria,viruses, and the like. In some embodiments the infectious pathogen to bedetected is anthrax, plague, or Ebola. However, it is not necessary forthe infectious agent to be known because the array of lipoparticles cancomprise lipoparticles that comprise different membrane proteins thatcan interact with different infectious pathogens.

As used herein, the term “agent” can refer to a chemical, compound, orinfectious pathogen that can cause sickness, disease, and/or death.

As used herein, the term “infectious pathogen” refers to amicro-organism (e.g. virus or bacteria) that can cause sickness,disease, and/or death. Examples of infectious pathogens include, but arenot limited to, HIV, Ebola, plague, E. Coli, anthrax, West Nile Virus,smallpox, chickenpox, monkey pox, hanta virus, SARS, tuberculosis,whooping cough, cholera, and the like.

Other examples of infectious pathogens, including their proposedreceptors, are provided in Table 6.

TABLE 6 Selected membrane proteins proven or proposed (in parentheses)to function as pathogen receptors. Membrane Protein Proven (or proposed)pathogen Usage Anthrax Toxin Receptor Anthrax Toxin binding Alpha andbeta dystroglycan Lassa Fever, LCMV hemorrhagic fever viruses CD44Shigella Mannose receptor (MRC1) Ricin toxin CFTR Salmonella entericaCCR5, CXCR4 HIV coreceptors, (smallpox, yersinia pestis) DC-SIGN,DC-SIGNR HIV, Ebola, Dengue, CMV, Candida

The present invention also relates to detecting any agent that can causesickness, disease, and/or death that may be present in a sample. Theseagents may be food-borne, water-borne, air-borne, present in fecalmaterial, and the like. The agent may be an organic compound, a peptide,a protein, a micro-organism (e.g. virus or bacterium), and the like. Itis not necessary that the agent be infectious (i.e. being capable ofbeing transmitted from one organism to another). The compound can beidentified as long as it is able to be bound by a protein or a membraneprotein that is part of a lipoparticle that is attached to a sensorsurface.

The present invention also relates to methods of determining thepresence of a substance comprising the steps of: contacting a samplewith an array of lipoparticles attached to a sensor surface, wherein thelipoparticles comprise membrane proteins that interact with thesubstance; and detecting an interaction with the array of lipoparticles,wherein the detection of the interaction indicates the presence of thesubstance.

An array of lipoparticles attached to a sensor surface can also be usedto detect a substance that is derived from saliva, blood, serum, urine,semen, vaginal secretions, cell lysate, cell supernatant, tissuehomogenate, food, water samples, and the like.

In some embodiments, an array of lipoparticles can be used to detect asubstance from a swarm of virus, which is, in some embodiments, obtainedfrom an infected individual.

The present invention also provides for the creation of an InfectiousDisease Receptor Biosensor (IDRB) using lipoparticles, such that knownmembrane proteins having functions known to be involved with infectiousdisease will be incorporated into the array.

As used herein the term “Infectious Disease Receptor Biosensor” refersto a sensor surface that is used in conjunction with a biosensor machineand/or system to detect infectious pathogens.

Such a tool can have extensive applications for diagnostics andbiomedical research. Using this tool, samples can be screened to detectmolecules which interact with known infectious disease related membraneproteins. As more infectious disease related proteins are discoveredthey can be incorporated into the Receptor Biosensor.

In some embodiments, a use of the IDRB with human clinical samples is todetect pathogens, toxins, and proteins. Pathogens tested can include,but are not limited to, both viruses and bacteria. Viruses can includesingle species as well as swarms that may use multiple receptors. Bothspiked control fluids and clinical samples from pathogen-infectedpatients can be tested.

A number of complex fluids (including serum, urine, and saliva) can beanalyzed to detect and quantify the presence of infectious agents inthese samples that bind to the membrane proteins represented on the cellsurface biosensor. Samples spiked with known ligands and antibodies canbe used as positive controls.

Such a focused receptor biosensor is useful for the generation of a kitdesigned for diagnostics where a sample is screened against knownmembrane proteins known to bind with soluble infectious agents.

The creation and screening of the IDRB will closely parallel the methodsdescribed for other biosensors described herein. First, lipoparticlescontaining known infectious disease related membrane proteins can beproduced. Second, these lipoparticles can be attached to a biosensorsurface. Finally, these biosensors can be screened using serum, urine,and saliva to determine the presence of infectious agents.

The present invention also provides for the creation of a Water QualityReceptor Biosensor (WQRB) using lipoparticles, such that known membraneproteins having functions known to be involved with contaminants to thewater supply can be incorporated into the array.

As used herein the term “Water Quality Receptor Biosensor” refers to asensor surface that is used in conjunction with a biosensor machineand/or system to detect water contaminants.

Such a tool can have extensive applications for diagnostics andbiomedical research. Using this tool, samples can be screened to detectmolecules that interact with known water contaminant related membraneproteins. As more water quality related membrane proteins are discoveredthey can be incorporated into the Water Quality Receptor Biosensor.Examples of water contaminants can be found Table 5, but any watercontaminant can be included.

The creation and screening of the WQRB will closely parallel the methodsdescribed for biosensors described herein. First, lipoparticles can beproduced containing known membrane proteins having functions known to beinvolved with contaminants to the water supply. Second, theselipoparticles can be attached to a biosensor surface. Finally, thesebiosensors can be screened using water samples to determine the presenceof contaminants. The result of this can be a tool to aid in the rapidtesting of multiple water samples for a large number of potentialcontaminants. One skilled in the art would also recognize that a similarsystem can also be used for detection of contaminants or components ofother liquids, beverages, foods, chemicals, or aqueous solutions.

The present invention also relates to methods of identifying aninhibitor of a substance comprising the steps of: contacting a substanceand a compound with an array of lipoparticles attached to a sensorsurface, wherein said substance can bind to said array of lipoparticlesin the absence of the compound; and detecting an interaction of saidsubstance with said array, wherein if an interaction is detected, thenthe compound does not inhibit the binding and if an interaction is notdetected then the compound inhibits the binding of the substance withthe lipoparticle.

Compounds that can be tested as inhibitors include, but are not limitedto, organic compounds, peptides, proteins, pharmaceuticals, nucleicacids, —e.g. RNA, DNA, oligonucleotides, double-stranded RNA, and thelike.

The present invention also relates to methods of identifying a bindingpartner for a substance comprising the steps of: a) contacting asubstance with an array of lipoparticles attached to a sensor surface;b) detecting an interaction of the substance with the array; and c)identifying the binding partner. In some embodiments, the bindingpartner is a membrane protein that can bind to the substance. In someembodiments, a substance is contacted with an array of lipoparticleswhere the composition of each lipoparticle is known. In some embodimentsthe composition of the lipoparticles is unknown, but the protein ofinterest can be determined through known cloning and sequencingtechniques. After contacting the substance with the array oflipoparticles, an interaction is detected by the sensor surface at aparticular position(s). The lipoparticles are identified and the bindingpartner present in the lipoparticle is identified as the binding partnerof the substance. The substance can be any substance which can bind to alipoparticle or a membrane protein present in the lipoparticle. Examplesinclude, but are not limited to an organic compound, chemical, peptide,protein, antibody, virus, bacteria, toxin, multiple proteins (i.e. morethan one), a pharmaceutical composition, a monoclonal antibody, achemokine, a cytokine, a secreted protein, a neurotransmitter, and thelike.

The present invention also relates to methods of determining aninteraction map of a substance comprising the steps of: a) contacting atleast one substance with an array of lipoparticles attached to a sensorsurface; b) measuring binding of said at least one substance with saidarray of lipoparticles; c) determining which lipoparticles bind with atleast one substance; and d) creating said interaction map based uponsaid binding.

As used herein, the term “interaction map” refers to the identificationof interactions that a particular substance makes with an array oflipoparticles. A substance is any substance that can interact with anarray of lipoparticles attached to a sensor surface, such as, but notlimited to, an organic compound, chemical, peptide, protein, antibody,virus, bacteria, toxin, multiple proteins (i.e. more than one), apharmaceutical composition, a monoclonal antibody, a chemokine, acytokine, a secreted protein, a neurotransmitter, and the like. Asubstance or a group of substances may interact with more than onemembrane protein and therefore with more than one lipoparticle presentin an array of lipoparticles. The “interaction map” would be acompilation of all the interactions that a substance or a group ofsubstances make. The interaction map may list only the positions on thearray that the substances interacts with, or in some embodiments, it maylist the proteins that are present at the interaction sites in thelipoparticles.

Interaction maps can be used for many purposes including, withoutlimitation, metabolism, toxicity, patient health, disease progression,clinical outcome, absorption, and the like. For example, serum may beobtained from a patient at different time points during the progressionof a disease. The serum can be contacted with an array of lipoparticlesattached to a sensor surface. An interaction map can be created fromeach time point and the changes that are seen during the diseaseprogression can be used to better treat the patient or to betterunderstand the disease and/or disease progression.

Interaction maps can also be used to understand toxicity of a drug. Adrug can be contacted with an array of lipoparticles attached to asensor surface and an interaction map can be created. If that drug isknown to be toxic, (i.e. have unwanted side effects), the interactionscan be listed as potentially being predictive of a drug's toxicity. Anovel drug can then be tested against an array of lipoparticles attachedto a sensor surface to create an interaction map. If the interaction mapof the novel drug is similar to the drug with the unwanted side effects,one of ordinary skill in the art could predict that the novel drug wouldalso have the unwanted side effects. The interaction maps can also beused in a similar way to map a patient's clinical outcome and patienthealth.

As is the case for all the sensor surfaces described herein, the sensorsurface can comprise a biosensor or a microfluidics device.

An interaction map can also be created for more than one substance.

The present invention also relates to methods for spottinglipoparticles, pseudotypes, or viruses in an array format onto a solidsurface without allowing the liquid to completely desiccate. In someembodiments, the spotting comprises including in the spotting mediumtrehalose, glycerol, sucrose, collagen, or gelatin. In some embodiments,the lipoparticles are dried or lyophilized.

The present invention also relates to methods of identifying a ligandfor, for example, a membrane protein, comprising the steps of: a)contacting a composition containing a potential ligand with an array oflipoparticles attached to a sensor surface; b) isolating said ligand;and c) determining identity of said ligand. An array of lipoparticlesattached to a sensor surface can also identify a ligand for thelipoparticle. For example, a membrane protein can be identified that isthought to interact with another substance, which can be any substancesuch as, but not limited to, an organic compound, chemical, peptide,protein, antibody, virus, bacteria, toxin, multiple proteins (i.e. morethan one), a pharmaceutical composition, a monoclonal antibody, achemokine, a cytokine, a secreted protein, a neurotransmitter, and thelike. An array of lipoparticles comprising the protein of interest iscontacted with at least one ligand or a mixture containing potentiallymultiple ligands. A ligand's binding is detected by the sensor surfaceand the ligand can then be isolated from the mixture by knowntechniques. The ligand can be isolated and identified by liquid or gaschromatography, mass spectrometry, antibody detection (i.e. WesternBlot, ELISA), northern blot, PCR, NMR, x-ray crystallography, and thelike. The ligand may be part of a library whose identity is alreadyknown and what is being identified is the interaction, or the ligand maybe unknown and part of a complex mixture. It should be noted thatbiosensors are now routinely being used in conjunction with massspectrometry to identify new ligands in eluted material (Nedelkov, etal. (2001), Biosensors & Bioelectronics, 16:1071-1078, Nedelkov, et al.(2001), Proteomics, 1:1441-1446, Williams, et al. (2000), Trends inBiotechnology, 18:45-48), however this has not been done withlipoparticles. One skilled in the art will recognize that futureapplications involving orphan receptors, de novo ligand design (below),and other applications may employ mass spectrometry.

In some embodiments the ligand is identified from saliva, blood, serum,urine, semen, vaginal secretions, cell lysate, cell supernatant, tissuehomogenate, food, water samples, and the like, or a combination thereof.

In some embodiments, the present invention also provides methods ofidentifying a binding partner of a membrane protein comprisingcontacting a lipoparticle, virus, or virus-like particle comprising themembrane protein with a library. In some embodiments, the librarycomprises more than one, more than 10, more than 50, more than 100, morethan 1,000, more than 5,000, more than 10,000, about 500, about 1,000,about 5,000, about 10,000 potential binding partners. In someembodiments, the method comprises detecting the binding of the bindingpartner to the membrane protein. In some embodiments, the library is aphage display library or ribosome display library. In some embodiments,the binding partner is a monoclonal antibody, a polyclonal antibody, anaffinity-purified polyclonal antibody, a Fab fragment derived from amonoclonal antibody, an immunoglobulin-fusion protein, a single-chainFv, an Fc-fusion protein, peptide, or polypeptide.

The present invention also relates to methods of ligand designcomprising the steps of: a) contacting at least one ligand with alipoparticle attached to a sensor surface; and b) identifying ligandsthat bind to said lipoparticle. In some embodiments, the methods furthercomprise modifying the ligand and repeating steps a and b modifying theligand until a ligand is identified that binds to the lipoparticle tosatisfy a user-defined criteria. In some embodiments, the lipoparticleto which the ligand binds comprises a membrane protein. In someembodiments, the user-defined criteria is a high binding constant.

Previous investigators have reported successfully using the BIACOREbiosensor for phage screening, although not with membrane proteins orlipoparticles (Malmborg, et al. (1996), Journal of ImmunologicalMethods, 198:51-57, Schatzlein, et al. (2001), J Control Release,74:357-362). The lipoparticle biosensor has predicted advantages overtraditional phage panning of wells or beads: 1) phage binding ismonitored in real time to better control specific binding, 2) bindingcan be monitored on control flow cells simultaneously for monitoring ofnon-specific binding, 3) elution can be monitored in real-time so onlythe fractions binding most strongly and specifically to the correctflow-cells need be collected, and 4) even weak affinities can bedetected with a biosensor so difficult receptor-ligand pairs still canbe identified. Several high affinity, short sequences of proteins thatbind CXCR4 have been identified by ourselves and others using techniquesthat did not involve lipoparticles (Crump, et al. (1997), EMBO J,16:6996-7007, Doranz, et al. (1997), J. Exp. Med., 186:1395-1400,Doranz, et al. (1999), J. Virol., 73:2752-2761, Murakami, et al. (1997),J. Exp. Med., 186:1389-1393), suggesting that high affinity peptides canbe identified when used with lipoparticles. It should be noted that thisapproach could also be used for a phage library displaying antibody (Fv)fragments, a popular commercial method for deriving human MAbs.

As used herein, the term “user-defined criteria” refers to a standard bywhich the iterative process is stopped. This could be, for example, howtight the binding of the ligand to the lipoparticle is. Any user-definedcriteria, however, can be used. In some embodiments, the ligand that isbeing identified and/or modified is a peptide, a protein, an antibody, anucleic acid, molecule, an oligonucleotide, —e.g., DNA, RNA,double-stranded RNA, and the like. In some embodiments, the ligand(s)are presented to the lipoparticles on a phage.

The present invention also relates to a composition of multiplexedbiosensors designed to recreate the cell surface proteins of a cell. Insome embodiments, the composition will be a biosensor chip that cancontain thousands of individually addressable membrane proteins. In someembodiments, the surface of the array comprises all membrane proteinsencoded by the human genome, and can detect binding of unlabeledmolecules (drugs or proteins) in real-time, and can quantify binding toeach receptor. Operationally, the end-user would simply insert a sampleof interest, such as human sera. One skilled in the art will recognizethat such a technology would allow the detection of interaction betweenany soluble protein with any integral membrane protein that the solubleprotein would naturally bind to. This could include finding unknownreceptors, ligands, or antibodies.

Kits

In some embodiments, the present invention relates to a disposablebiosensor component that can be used to identify an infectious pathogenor other disease or sickness causing agent. In some embodiments thedisposable biosensor component is used to detect or identify Ebola,anthrax, plague, smallpox, SARS caronavirus, West Nile Virus, and thelike. In some embodiments, the disposable biosensor component will allowfor screening of samples for infectious pathogens or disease or sicknesscausing agents that interact with membrane proteins on the biosensor.

Lipoparticles Comprising Ion Channels and Methods Using the Same

In the present invention, the lipoparticles comprise ion channels ortransporter proteins and are used to measure function. When makinglipoparticles, contaminants of unwanted proteins may also be included inthe lipoparticle. In some embodiments, to avoid the contaminatingproteins from having undesired effects on how one uses thelipoparticles, the lipoparticles can be contacted with contaminatingprotein inhibiting toxins and/or ionophores to inhibit the contaminatingproteins. The contaminating protein inhibiting toxin and/or ionophore isselected so that it does not inhibit or affect the protein(s) that aredesired to be present in the lipoparticle.

In some embodiments, a virus, lipoparticle, or virus-like particle canbe used to measure ion channel or transporter protein function. Forexample, viruses such as influenza contain ion channels (e.g. M2) andthe methods described herein can be used assess the function of virallyencoded ion channels and transporter proteins in a virus or virus-likeparticle.

In some embodiments, the ion channel protein is a neurotransmitterreceptor.

The lipoparticles of the present invention can also be modified suchthat the modified lipoparticle can be used to detect ion channel proteinfunction or transporter protein function. The modifications can be anymodification that results in a parameter that can be measured,quantified, visualized, and the like. Examples of modifications that canbe made to lipoparticles include, but are not limited to, modifying themembrane lipid composition, modulating the fluorescent dye content ofthe lipoparticle (e.g. adding fluorescent dyes), modulating the watercontent of the lipoparticle, and modulating the ion content of thelipoparticle. In some embodiments, a change in the detectable agent canindicate an increase in protein function. In some embodiments, a changein the detectable agent indicates a decrease in protein function.

The fluorescent dyes can be used to monitor, visualize and/or measureprotein function. For example, when an ion channel or transporterprotein is activated, the dye can either flow into or out of thelipoparticle, which would indicate, in some embodiments, that proteinsare functioning. Similarly, when an ion channel or transporter proteinis activated, an ion or molecule can either flow into or out of thelipoparticle and interact with a dye either inside or outside or in themembrane of the lipoparticle, which would indicate, in some embodiments,that proteins are functioning. The amount of dye and the fluorescentsignal that is generated can be used to determine the level of activityof the membrane protein.

The present invention also provides for methods to determine membraneprotein function using a lipoparticle comprising the membrane protein.In some embodiments, the lipoparticles comprises an ion channel ortransporter protein and a detectable agent. The detectable agent can beany agent that can be detected by any means. For example, the agent canbe detected using fluorescence, ultra-violet light, or visual light.Examples of detectable agents include, without limitation,voltage-sensitive fluorescent dyes and ion-sensitive fluorescent dyes.

As used herein, the term “voltage-sensitive fluorescent dye” refers to adye that fluoresces or changes its fluorescent properties in response toa change in voltage.

As used herein, the term “ion-sensitive fluorescent dye” refers to a dyethat fluoresces or changes its fluorescent properties in response to achange in ion concentration.

Examples of ion-sensitive and voltage-sensitive fluorescent dyesinclude, but are not limited to, di-4-ANEPPS(C₂₈H₃₆N₂O₃S), di-8-ANEPPS,rhodamine 421, oxonol VI, JC-1, DiSC3(5), CC2-DMPE, DiSBAC2(3),DiSBAC4(3), VABSC-1, and the like (Molecular Probes, Inc.).

The choice of which detectable agent is used is not essential anddepends on the use of the lipoparticle. A plethora of fluorescent dyesand probes exist for detecting the function of ion channels, rangingfrom ion-specific dyes to dyes that respond to changes in membranepotential (Molecular Probes Handbook (2002)). To choose a probe, definedcriteria exist by which dyes may be chosen, these include but are notlimited to: easy to incorporate into pre-formed lipoparticles(encapsulation not necessary); versatility in detecting the function ofa range of ion channels (K⁺, Na⁺, Cl⁻, H⁺, Ca⁺²); high signal-to-noiseand sensitivity to voltage changes caused by ion channel gating; orratiometric measurement of fluorescent wavelengths, which provides aninternal standard for a more stable baseline independent of artifactssuch as leakage and loading. Ratiometric measurements can increasesignal:noise values over 10-fold by better discrimination between boundand unbound forms of the dye (Molecular Probes Handbook (2002), Montana,et al. (1989), Biochemistry, 28:4536-4539).

In some embodiments, membrane potential fluorescent dyes are used. Suchdyes were first used in the early 1970s, many being identified during asystematic search by Cohen and Salzberg (Cohen, et al. (1978), RevPhysiol Biochem Pharmacol, 83:35-88). Fluorescent dyes of membranepotential are classified as either “slow” or “fast” dyes, based on theirmechanism of action (Haugland (2003), (2002), Plasek, et al. (1996), Jof Photochemistry and Photobiology, 33:101-124, Smith (1990), BiochimBiophys Acta, 1016:1-28). Fast-response dyes respond to a change inmembrane potential with a change in their electronic structure (spatialrearrangement of valence electrons) and, consequently, theirfluorescence properties. Their optical response is sufficiently fast todetect transient (millisecond) potential changes, but the magnitude oftheir fluorescence change is relatively small, approximately 2-10% per100 mV. Slow-response dyes respond to a change in membrane potentialwith a transmembrane redistribution that is accompanied by afluorescence change. Their optical response is slower than fast probes(seconds to minutes), but the magnitude of their fluorescence change isgreater, up to 100% per 100 mV.

Of the more than two dozen different membrane potential dyes available,examples include, but are not limited to, the ANEPPS dyes, di-4-ANEPPSand di-8-ANEPPS, with di-4-ANEPPS being preferred. Both ANEPPS dyes haveidentical fluorophores, exhibit good photostability, low toxicity, and afairly uniform 10% per 100 mV changes in fluorescence intensity(Molecular Probes Handbook (2002), Rohr, et al. (1994), BiophysicalJournal, 67:1301-1315). Due to its longer alkyl chains, di-8-ANEPPS isbetter retained in the membrane, slightly more photostable, and lessphototoxic, but also more difficult to work with. Both ANEPPS dyes areessentially nonfluorescent in aqueous solutions but haveabsorption/emission maxima of 467/631 nm when bound to lipid membranes.Di-4-ANEPPS can be incorporated into lipoparticles (see, for example,FIG. 9). Further, the ANEPPS dyes can be measured ratiometrically,responding to increases in membrane potential with a decrease influorescence excited at approximately 440 nm and an increase influorescence excited at 530 nm. Ratiometric measurements between +120 mVand −120 mV are linearly responsive to membrane potential using ANEPPSdyes.

A number of alternative dyes are also available that can measuremembrane potential, ((2002), Plasek, et al. (1996), J of Photochemistryand Photobiology, 33:101-124, Smith (1990), Biochim Biophys Acta,1016:1-28). These dyes include but are not limited to the following.

Oxonol VI. Lipophilic anionic dyes such as the oxonols can detectrelatively large changes in membrane potential which occur over periodsof several minutes. In addition to the traditional use of oxonol todetect absolute changes in fluorescence, a ratiometric method has beendeveloped using a fluorescence resonance energy transfer mechanism basedon reactivity between oxonol and fluorescently labeled lipids (Gonzalez,et al. (1995), Biophysical Journal, 69:1272-1280). This approachreportedly can sense fast potential changes with fluorescence changesthat exceed 50% per 100 mV

JC-1. JC-1 is a carbocyanine fluorescent dye that forms aggregates upondepolarization and can be measured ratiometrically. Aggregation withinthe confined membrane interior results in decreased fluorescence andmaximal emission shifts from 527 nm to 590 nm. The dye has been usedsuccessfully in many experiments, but is noted by some to exhibitunacceptable variability.

DiSC3(5). For many years, DiSC3(5) was considered to be the dye ofchoice for membrane potential assays due its high sensitivity, 50-80%per 100 mV (the highest of all cyanine dyes). This high signal isunavoidably related to the dye's high accumulation in cells, and thus toits high toxicity, but the toxicity should not effect signal withinlipoparticles. Depending on the environment of use, DiSC3(5) may beassayed ratiometrically, increasing signal-to-noise about 10-fold.

Rhodamine 421. RH 421 has yielded the most sensitive response recordedfor a fast potentiometric probe, greater than 20% fluorescence changeper 100 mV. The optimal excitation and emission from RH 421 is dependenton its environment.

The properties of some fluorescent dyes that can be used to measuremembrane potential are listed in Table 7. Other detectable agentsinclude, but are not limited to fluorescent probes (e.g. fluorescentproteins, fluorescent amino acids, or fluorescent lipids). In someembodiments, the detectable agents can be used to make a ratiometricmeasurement to determine the function of the membrane protein.

TABLE 7 Fluorescent dyes are listed. Dyes were selected from more thantwo dozen fluorescent dyes that are responsive to membrane potential(Molecular Probes Handbook (2002),, Rohr, et al. (1994), BiophysicalJournal, 67: 1301-1315). Category Dye RatiometricAdvantages/Disadvantages Fast Di-4-ANEPPS yes Fast response (msec)/Di-8-ANEPPS yes Low signal (10%/100 mV) Rhodamine 421 no Slow Oxonol VIyes Slow response (sec-min)/ JC-1 yes High signal (100%/100 mV) DiSC3(5)yes Slow dyes noted for variability of response

As used herein the term “ratiometric measurement” refers to the ratio ofat least two measurements that are used to create a ratio. For example,di-4-ANEPPS responds to an increase in membrane potential with adecrease in fluorescence excited at approximately 440 nm and an increasein fluorescence excited at 530 nm. By measuring the emission wavelengthat 630 nm, the 440/630 response can be divided by the 530/630 responseto produce a ratio measurement.

As with all methods described herein, a computer can be used tocalculate and measure the detectable agent, and calculate and determinethe ratiometric measurement, and the like.

When using fluorescent detectable agents, one can measure the functionof the membrane protein by resonance energy transfer (FRET). Resonanceenergy transfer is well known to one of ordinary skill in the art. Forexample, two separate fluorescent species can interact and they generatea distinct and different fluorescent signal. The distinct signal can beused to determine the function of the membrane protein.

In some embodiments, the present invention provides for methods ofidentifying inhibitors or stimulators of proteins (e.g. GCPRs, ionchannels, transporter proteins and the like) comprising measuringchanges in detectable agents. A lipoparticle comprising a membraneprotein (e.g. ion channel or transporter protein) and a detectable agentwill generate a detectable signal when the protein is active orinactive. The lipoparticle can be contacted with a test compound and achange in the detectable signal indicates that the compound is either aninhibitor or a stimulator. The change in the detectable signal could bea complete inhibition of the detectable signal (e.g. the signal is nolonger detectable) or the detectable signal may be reduced. In someembodiments, the detectable signal may increase. Examples of testcompounds include but are not limited to, proteins, peptides, aminoacids, organic molecules, antibodies, nucleic acids, inorganiccompounds, and the like.

The present invention also provides for methods for identifyinginhibitors of a known stimulator of a membrane proteins (e.g. GCPRs, ionchannels, or transporter proteins) within a lipoparticle comprisingcontacting the lipoparticle with the stimulator and the potentialinhibitor. In some embodiments, the potential inhibitor and stimulatorare contacted concurrently. In some embodiments, the potential inhibitoris contacted with lipoparticle before or after the stimulator iscontacted with the lipoparticle. This method can be used, for example,to test antagonists of known ligands of GCPRs, ion channels, andneurotransmitters. In some embodiments, instead of an inhibitor, anagonist is used to determine what effect the agonist has on the functionof the membrane protein.

As used herein, the term “inhibitor” refers to a compound, peptide, orprotein that inhibits the function of a protein. In some embodiments,the inhibitor is an antibody or fragment thereof.

Toxins are known to bind membrane proteins. Some toxins can only bindwhen the membrane protein is in a certain conformation or active state.Therefore, in some embodiments, the present invention provides methodsof confirming membrane protein conformation by binding a toxin to alipoparticle containing an ion channel or membrane protein comprisingcontacting the lipoparticle with the toxin. In some embodiments, theability of the toxin to bind to the membrane protein indicates thestructure or active state of the ion channel. The binding status of thetoxin can also be used to determine that the membrane protein hasproperly folded and presented in the correct conformation on the surfaceof the lipoparticle. Therefore, this can be used in some embodiments asa quality control test to determine the structure and folding of amembrane protein in a lipoparticle.

The present invention also provides for methods of measuring membraneprotein function comprising the steps of microinjecting lipoparticles toa location and measuring the function of the membrane protein. Thelocation can be, for example, an intracellular compartment, an organelle(e.g. mitochondria), the cell surface, gap junctions, or a synapse. Oncethe lipoparticle is microinjected into its location, the lipoparticlecan be used to detect membrane protein function. The lipoparticle can beused to detect the function of the membrane proteins within thelipoparticle and the lipoparticle can also be used to detect changes inthe environment (e.g. ion concentration) of its surroundings. Forexample, if the ion concentration of the environment in which thelipoparticle has been injected into changes, a lipoparticle comprisingan ion channel and a fluorescent dye can be used to detect this change.The change in ion concentration can, in some embodiments, open the ionchannel allowing the signal generated by the fluorescent dye toincrease, or in some embodiments, decrease thereby indicating a changein the location. The lipoparticle comprising an ion channel can be usedto detect the change in ion concentration or membrane potential. As withall the methods described herein, in some embodiments, the measuredfunction of the ion channel is the absolute level. This can involvecalibrating the lipoparticles to a calibration standard. Examples of acalibration standard include, but are not limited to, ionophores. Whenused with known molar amounts of a specific ion (e.g. potassium), theionophore allows the ion to permeabilize the membrane, as measured by a100% signal. By knowing the ion concentration and using the Nernstequation, the 100% signal can be converted to millivolts of membranepotential. The signal measured experimentally can then be calibrated toabsolute units (e.g. millivolts).

As used herein, the term “absolute level” refers to the quantity of asubstance as measured in units that represent the level of thesubstance, independent of experimental measurement. Units of absolutelevels can be in molar or millivolts. For example, the membranepotential of a typical living cell in absolute units is approximately−70 mV.

The present invention also provides for kits for assessing the functionof an ion channel or transporter, wherein the kit comprises alipoparticle comprising a desired membrane protein and a protocol forassaying function.

The present invention facilitates the detection of ion channel functionwithin a nano-scale. The ability to sense ion channel function within anano-scale sensor using lipoparticles has many applications, including,for example, microfluidic drug screening, and subcellular detection.

The benefits of microfluidics, in turn, include miniaturization,integration of multiple processes, automation, reduced labor cost,reduced reagent requirements, multiplexed detection, and higher speed.Cells, however, cannot be used within many microfluidic devices becauseof their size and environmental requirements for viability. Atcommercial use, lipoparticles can be used for high throughput testing ofdrug candidates for inhibition of ion channels using microfluidicdevices. In the simplest case, an array of several hundred lipoparticleswill be created using multi-well plates (e.g. 384-well). In moresophisticated applications, lipoparticles can be attached to sensorswith microfluidic flow channels (e.g. BIACORE, CALIPER systems) thatallow a continuous flow detection system. These systems are capable ofdetecting interactions with more than one target at a time(multiplexing), making screening even more efficient. Other devices thatcan be incorporated with lipoparticles to measure function or activityinclude, but are not limited to a Lab-on-a-Chip™, a 96-well plate, a384-well plate, a 1536-well plate, a glass slide, a plastic slide, anoptical fiber, a flow cytometer, a microscope, a fluorometer, aspectrometer, or a CCD camera.

Nanometer-sized sensors of ions and voltage can also be used to probesubcellular structures during physiological responses. For example,neurons can be monitored under conditions that they are activated.Moreover, the probes that can be constructed are not limited to sensingchanges, but can be calibrated to detect absolute levels of ions andvoltage, allowing local measurements of important, but inaccessible,structures.

The lipoparticles of the present invention can be readily tested todetermine if the membrane protein is functional and detectable asdescribed herein. There are a number of reasons that the membraneprotein (e.g. ion channel or transporter protein) function cannot bedetected. One such reason is that the interior is not large enough toallow for detection of functionality. Therefore, in some embodiments, atleast two lipoparticles can be fused together to increase the interiorvolume of the lipoparticle. One can also increase the size of thelipoparticle by increasing the lipid concentration or by fusing it witha liposome. One of skill in the art would readily know how to fuse atleast two particles together (see, for example, Sparacio et al.,Virology. 2000 271:248-52 and Sparacio et al. Virology. 2002 Mar. 15;294(2):305-11). In some embodiments, at least 3, at least 4, or at least5 lipoparticles are fused together. In some embodiments, the fusedlipoparticles are isolated and used based on their size, which can bedone using methods such as, for example, fractionation, sedimentation,centrifugation, column chromatography, HPLC, FPLC, and the like.However, any method to isolate a lipoparticle of a particular size ordetermine the size of a lipoparticle can be used.

Antibody Production

Lipoparticles provide a novel mechanism for the presentation of antigensto the immune system, with unprecedented control over the antigen beingpresented.

In some embodiments, lipoparticles are used for the direct presentationof membrane proteins for antibody generation. Antibodies are now in usethroughout the biotechnology industry as therapeutics, diagnostics, andR&D reagents, and are an inherent product of vaccination.

A lipoparticle allows the stable presentation of structurally intactmembrane proteins within a particulate format that is suitable forantigen presentation because the structure of complex membrane proteinscan be maintained using the lipoparticle. In some embodiments, thepresent invention provides for methods of using lipoparticles comprisinga protein of interest as an antigenic composition for production ofantibodies that specifically bind with the membrane protein.

As used herein, the phrase “protein of interest” refers to a protein forwhich antibody screening and/or antibody generation is desired. Theprotein can be any protein and can include, for example, intracellularproteins, membrane proteins (e.g. ion channels), receptors (e.g. Gprotein-coupled receptors), membrane proteins from infectious pathogens(e.g. membrane proteins from viruses), and the like. A “protein ofinterest” can also refer to a protein that is to be included or isincluded in a lipoparticle. In some embodiments, the “protein ofinterest” is a membrane protein.

In some embodiments, the antibodies produced can bind with the proteinin its native structure, and thus the present invention provides methodsfor producing antibodies that can, for example, inhibit protein functionby steric blocking and/or antibodies that can affect protein function byallosteric effect. The production of lipoparticles using viral vectorscan allow lipoparticles to be produced for antibody purposes usingnon-human (e.g. mouse) cells for incorporation of human membraneproteins.

Examples of non-human cell types include, but are not limited to, mousecells, goat cells, rabbit cells, sheep cells, donkey cells, quail cells,and the like. The advantage of using non-human cell types for theproduction of antibodies is that, if the lipoparticle produced from thenon-human cell type is injected into an organism that is the same fromwhich the non-human cell type was derived, the immune response generatedby the organism should be specifically directed at the cellular proteinof interest that is not native to the non-human cell type. This mayenable the generation of antibodies that are more specific for thecellular protein of interest rather than the other components of theimmunogen (e.g. the lipoparticle membrane and other proteins).Lipoparticles can also be produced from immortalized cell lines, stemcells, primary cells (e.g. limited life span), and hybridomas.

Therefore, in some embodiments, the present invention provides forimmunogens comprising a lipoparticle, wherein the lipoparticle comprisesa cellular protein of interest. In some embodiments, immunogens can bemodified for a number of reasons, for example, to increase theimmunogenicity of the lipoparticle. The modification can comprisemodifying either the lipoparticle, the cellular protein of interest, ora combination thereof. The cellular protein of interest can be modified,for example, by modifying the amino acid sequence (e.g., to render theprotein more immunogenic or antigenic).

Modifying the amino acid sequence of the cellular protein of interestcan include nonsense mutations, missense mutations, conservativesubstitutions, non-conservative substitutions, deletions of one or moreamino acids, insertions of one or more amino acids, and the like. Thecellular protein of interest can also be modified by creating a chimericprotein containing a portion of the cellular protein of interest and aportion of another polypeptide. A chimeric protein can also be referredto as a “fusion protein.” As used herein, the term “portion” refers toeither the whole protein or a fragment of the entire protein. In someembodiments, the chimeric protein comprises a non-fluorescent proteinand a fluorescent protein.

The modification of the cellular protein of interest can also affect thefunction of the protein. The modification can create a constitutivelyactivated protein or an inactive protein. The activity of the proteinneed not be present in the lipoparticle, rather whether the modificationaffects the activity of the protein can also be determined in a cellularenvironment, in a test tube and the like.

Modifications can also include adding or removing targeting signals onthe polypeptide to ensure that the cellular protein of interest istargeted to the membrane of the cell and is contained in thelipoparticle that is produced from the cell. See, U.S. ProvisionalApplication Ser. No. 60/491,477.

Modifications to the lipoparticle to increase immunogenicity of theantigen can also comprise any modification known to those of skill inthe art. These modifications can comprise, for example, a solubleprotein bound to the cellular protein of interest in the lipoparticle.The soluble protein can be bound to the cellular protein of interest,for example, by cross-linking the soluble protein to the cellularprotein. The soluble protein can also be tethered to the lipoparticle.The tether can be, for example, a fusion of a soluble protein andpolyethylene glycol (PEG), a fusion of the soluble protein to atransmembrane protein, or a fusion of the soluble protein to a GPIanchor. For example, the soluble chemokine RANTES can be cross-linked ortethered to its cognate receptor CCR5. Other ligands could also work,for example MIP1alpha or MIP1beta. In addition, other receptors couldwork, such as, without limiting the number of receptors possible, CCR2,CCR3, or CXCR4.

As used herein, the term “soluble protein” refers to a protein that issoluble. In some embodiments the soluble protein is the protein ofinterest. In some embodiments, the soluble protein is a ligand or abinding partner of the protein of interest.

Lipoparticles engineered to contain both a receptor and a tetheredligand offer possibilities to immobilize the receptor in their engagedconformation. For example, the CCR5/MIP1α receptor-ligand system can belinked to the lipoparticle such that the CCR5 is incorporated into themembrane, retaining its secondary structure, and MIP1α is tethered tothe distal end of a phospholipid-poly(ethylene glycol) (PL-PEG). Thisallows the lipoparticle to present the receptor bound to the ligandwithout the worry of diffusion of the ligand. Mabs could be generatedagainst the activated receptor which may have a different conformationthan the non-activated receptor. PL-PEG-Biotin may also be linked to theCCR5 expressing lipoparticle. Subsequent contact with a fusion proteinmade of avidin and MIP1α will essentially link the MIP1α to the membraneby the biotin avidin complex formation.

The creation of a fusion protein consisting of both the receptor and theligand may serve as an alternative to tethering, incubating in ligand,or incubating in ligand and cross-linking. In this case a ligand will be“connected” to the extracellular portion of the receptor by expressing afusion protein with the sequence of the ligand on the end of thesequence of the receptor. For example, the chemokine ligand RANTES canbe expressed at the N-terminus of CCR5, its receptor. Different fusionproteins with peptide spacers can be constructed to allow the ligandenough room to a) adopt an appropriate conformation and b) interact andbind with the receptor.

The antibodies generated by tethering or cross-linking can, in someembodiments, specifically recognize receptors which are bound by theirligand. Such antibodies can be used, for example, to determine the stateof a given receptor or tested for their ability to alter the stabilityof the receptor-ligand interaction. The antibody may cause a ligand tohave a stronger interaction with the receptor and bind for a longer timeresulting in more signaling. This has the potential for therapeutic usein cases where signaling through a particular receptor is insufficient.Furthermore, these antibodies, in some embodiments, can have thecapacity to recognize a complex, providing a research tool for showinghow and where the complex forms, diagnostic tools for seeing if thecomplex forms, and therapeutic tools for targeting the complex.

In some embodiments, the cellular protein of interest may be a proteinthat is a naturally expressed membrane protein from a specific cell orcell type. As used herein, the term “naturally expressed” refers to aprotein that is expressed by the cell without the introduction of anexogenous polynucleotide encoding for a protein. Therefore, antibodiescan be produced against naturally expressed proteins that can becaptured by a lipoparticle when produced from that the cell containingthe naturally expressed protein.

The immunogenic compositions can also further comprise at least oneimmunostimulatory component besides the lipoparticle comprising thecellular protein of interest. In some embodiments, the immunogenicand/or antigenic composition can comprising at least one, at least two,at least three, at least four, at least five, or at least 10immunostimulatory components. As used herein, the term“immunostimulatory component” refers to a component that facilitatesgeneration of an immune response. The immunostimulatory component canbe, for example, DNA, RNA, oligonucleotide, adenovirus, an adjuvant, oranother polypeptide. Adjuvants, however can alter the structure ofcellular protein of interest contained in the lipoparticle and thus, insome embodiments, an adjuvant is used that does not destroy thestructure of the cellular protein of interest.

As used herein the term “adjuvants” refers to compounds that areinsoluble or undegradable substances that non-specifically enhance theimmune response to an antigen by promoting nonspecific inflammation inorder to recruit mononuclear phagocytes to the site of immunization. Insome embodiments, adjuvants incorporate two components. The firstcomponent can form a deposit to protect the antigen from catabolism.Mineral oils or aluminum hydroxide precipitates are typically used, butliposomes and synthetic surfactants have also worked well (R Hunter, etal, (1981), J Immunology, 127:1244-50). The second component can be anonspecific stimulant that activates antigen processing cells to secreteincreased levels of lymphokines and cause a local inflammatory reaction.Heat-killed bacteria (Bordetella pertussis or Mycobacteriumtuberculosis) or lipopolysaccharide are non-limiting examples ofnonspecific stimulants.

The use of adjuvants can have a marked effect on the ability to elicitan immune response, but each adjuvant has unique characteristics thatmay or may not be useful for immunization with lipoparticles. CompleteFreund's adjuvant, for example, has resulted in only linear epitope Mabsin prior experiments, suggesting that its composition is not conducivefor retention of receptor conformation. Ribi adjuvant, on the otherhand, has proven to be capable of maintaining the conformationalstructure of complex proteins during immune stimulation. Similarly,TiterMax (CytRx, Norcross, Ga.) is designed specifically to retainprotein structure for use with Mab immunization, suggesting that it toowould not denature protein conformation during immunization.

The immunostimulatory component can also comprise a chemokine or acytokine, which can be used to enhance the immunogenicity of thelipoparticle. Examples of chemokines include, but are not limited to,RANTES, MIP1a, MIP1b, and the like. Examples of cytokine include, butare not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, GM-CSF and the like.

Membrane bound receptors may alter their extracellular epitopes whenactivated by their ligand and lipoparticles can be used to generateantibodies that identify epitopes specific to membrane proteins that arebound by their ligand. These epitopes can be, for example, the membraneprotein, the ligand, a combination of both, or a new epitope on eitherthat is exposed by conformational changes induced by ligand binding.

Intracellular proteins can also influence the extracellular structure ofmembrane proteins or receptors. For example, it is has been establishedthat many GPCRs can bind agonists with high affinity when cytosolicG-proteins are simultaneously bound, but have only weak affinity forthese same agonists when G-proteins are not bound. With GPCR crystalstructures extremely difficult to obtain, modeling of the structuralchanges of GPCRs upon G-protein binding remains poorly defined.Antibodies that can detect such structural changes could be used todetect and measure active and non-active conformations of the desiredreceptor within cells, as well as help determine the intrinsic activityof GPCRs. Therefore, in some embodiments, antibodies can be generatedagainst membrane proteins that can differentiate between a protein boundto a cytosolic protein or not bound to a cytosolic protein. Examples ofGPCRs include, but are not limited to, ORF74, CXCR2, and β-adrenergicreceptor. Therefore, in some embodiments, the immunogens of the presentinvention can also include one or more binding substances that bind tothe cellular protein of interest. Examples of binding substancesinclude, for example, a ligand, G-protein, a kinase, a phosphatase, anantibody, a peptide, small organic molecule, and the like. The bindingsubstance can change the conformation of the receptor. In one way, bymere fact of binding a different structure has been created (thereceptor+ligand vs. the receptor alone). Some antibodies recognizeepitopes that are formed by both the receptor and ligand. The second isallosteric—the ligand binds and changes the shape of the receptor. Thereare antibodies that recognize such epitopes (e.g. the MAb 17b recognizesan epitope within HIV-1 gp120 that is exposed only after binding of thereceptor CD4) (Hoffman, et al. (1999), Proc. Natl. Acad. Sci. USA,96:6359-6364). In some embodiments at least one, at least two, at leastthree, at least four, at least 5, or at least ten binding substances canbe used.

Mabs can be used to characterize the protein expression patterns ofGPCRs in cells that have previously only been detectable functionally orthrough RNA expression analysis.

As used herein, the term “antibody” is meant to refer to complete,intact immunoglobulin molecules as well as fragments thereof including,without limitation, Fab fragments and F(ab)₂ fragments thereof, animmunoglobulin-fusion protein, a single-chain Fv, and an Fc-fusionprotein. Complete, intact immunoglobulin molecules include, but are notlimited to, polyclonal antibodies, monoclonal antibodies such as murinemonoclonal antibodies, rabbit antibodies, goat antibodies, donkeyantibodies, horse antibodies, chicken antibodies, human antibodies,chimeric antibodies and humanized antibodies. Antibodies that bind to anepitope on the cellular protein are also useful to isolate and purifythat protein from both natural sources or recombinant expression systemsusing well known techniques such as affinity chromatography. Suchantibodies are useful to detect the presence of such protein in a sampleand to determine if cells are expressing the protein.

A number of important classes of both multi-spanning and single-spanningproteins form homo- or hetero-oligomers (i.e. multimers) in a lipidmembrane, including ion channels, integrins, Tyr-kinases such as the EGFreceptor, some GPCRs, Hepatitis C E1-E2, and HIV Envelope. Mabs thatrecognize multimeric complexes could be especially useful in identifyingthe importance of multimerization to signaling, subcellular locations inwhich multimers form, and the importance of multimerization for somediseases. The domains that mediate this oligomerization are oftenlocated within cytoplasmic or transmembrane regions. Otheroligomerization domains are located extracellularly, often near thetransmembrane domain, but are dependent on protein structures within thetransmembrane or cytoplasmic domains. Lipoparticles can be useful forthe presentation of such proteins since the proteins can be concentratedon the surface of the lipoparticle, hence stabilizing multimericstructures that might otherwise be short-lived or interact with lowaffinity.

Mabs can also have the additional ability to detect oligomers.Functional uses of these Mabs can include channel or receptor blocking,inhibition, and activation. Specifically, in the case of Kv1.3 (apotassium channel), the Mab can be used for immunosupression. Antibodiesto DC-SIGN (Dendritic Cell-Specific, ICAM-3 Grabbing Non-integrin) canbe used in the rhesus macaque model to determine if they impact sexualtransmission of either SIV or SHIV animal models. Mabs to a ErbB2/ErbB4heterodimer may help identify particularly aggressive forms of cancer,and help direct appropriate treatment.

Therefore, in some embodiments, the antibodies are specific to anepitope that is specific to the quaternary structure of a protein. Thecellular protein of interest having the quaternary structure can be theresult of a cellular protein or a membrane protein forming an oligomericstructure. In some embodiments, the oligomeric structure comprises ahomo-oligomer. In some embodiments, the oligomeric structure comprises ahetero-oligomer. The oligomeric structure can be, for example, a dimer,a trimer, tetramer, or higher order oligomer. The higher order oligomermay comprise at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 members.

Antibodies can also be generated against less complex membrane boundproteins and single transmembrane proteins, where conformation may beless important. The antibodies directed against less complex membraneproteins, as well as the other antibodies discussed herein can be usedfor many applications including, but not limited to, Western blotanalysis, immunocytochemistry, immunohistochemistry, signal blocking orinhibition, and activation of pathways. For example, a CD4 antibodycould be used to crosslink CD4 and inhibit the immune response for organtransplantation or to model the clinical symptoms of AIDS. Monoclonalantibodies to Resistin could be administered to Type 2 diabetes patientsto improve their blood sugar and insulin action.

The generation of antibodies and the protein structures of complete,intact antibodies, Fab fragments and F(ab)₂ fragments and theorganization of the genetic sequences that encode such molecules arewell known and are described, for example, in Harlow, E. and D. Lane(1988) ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. which is incorporated herein by reference. Forexample, to produce monoclonal antibodies the lipoparticle is injectedinto mice. The spleen of the mouse is removed, the spleen cells areisolated and fused with immortalized mouse cells. The hybrid cells, orhybridomas, are cultured and those cells which secrete antibodies areselected. The antibodies are analyzed and, if found to specifically bindto the cellular protein, the hybridoma that produces them is isolatedand expanded in culture to produce a continuous supply of antibodies.

The present invention also provides methods of producing polyclonalantibodies and/or vaccinating against a membrane protein. Antibodies canbe produced against membrane proteins by introducing lipoparticlescontaining the membrane protein into an animal to produce antibodies.The antibodies are then isolated by techniques well known to those ofskill in the art and screened against the membrane protein to determineif the antibodies recognize the membrane protein. The antibodies can bescreened against a recombinant form of the protein or be screenedagainst samples that are known to contain the membrane protein. Theantibodies can also be compared to other known antibodies that recognizethe membrane protein to determine that the antibodies are recognizingthe proper membrane protein. In some embodiments, the antibodiesgenerated are specific for the membrane protein. As used herein, theterm “specific for the membrane protein” refers to an antibody thatrecognizes one membrane protein and no other membrane protein.

The present invention can also be employed to generate antibodiesagainst proteins which are difficult to express or which undergopost-translational modifications which require other genes present inthe natural state.

Capture of naturally expressed membrane proteins can result inpopulations of membrane proteins that better represent native membraneprotein structure. Some proteins are modified after translation, andthis modification may depend on factors within the cell. For example,CCR5 is sulfated on Tyrosine 11 and this sulfation is known to alter itsstructure and make it more competent for interaction with HIV-1 Envelopeproteins (Farzan, et al. (1999), Cell, 96:667-76). Similarly, CXCR4 isglycosylated, and if this glycosylation is removed, additionalstructures of CXCR4 are exposed (Chabot, et al. (2000), J Virol,74:4404-13). Different cell types are known to have differentconformations of the CXCR4 membrane protein. Other cell types can beinduced (e.g. with hormones, growth factors, cytokines, or chemicals) todifferentiate or change, often resulting in a change in the membraneproteins at the surface of the cell. Different cell types can alsocontain different transcriptional splice variants of the same gene. Forexample, CCR2 has two cell-type specific splice variants, CCR2a andCCR2b, which have differences in their C-terminus.

The generation of lipoparticles from natural cell sources can circumventthe difficulties that arise from using non-natural cell sources (e.g. nopost-translational modifications). Post-translational modification andinteraction with other proteins from the cells is insured by using thenatural cells, which should increase the likelihood of generating anantibody against a proper epitope.

In some embodiments, antibodies can be generated that are specific to anindividual cancer patient's tumor. Lipoparticles can be used to isolatethe membrane proteins of tumors cells and present them for antibodyproduction. This can be accomplished, for example, by taking a biopsy ofa tumor and isolating cancerous cells. These cells can be grown in cellculture and an aliquot of the culture can be used to generatelipoparticles as described herein. Antibodies can then be producedagainst the membrane proteins of the tumor cells. The antibodies canthen be screened against the cancer cells in culture and the effects oncell growth and death can be observed. In some embodiments, antibodiesthat trigger cell death can be used as a drug to treat the patient'stumors. Multiple iterations of this procedure can be performed toidentify proteins that are often expressed and tumors can be classifiedby the membrane proteins that they are expressing. In some embodiments,this information can be used to improve the treatment of cancer. Thisprocedure can also be modified to screen membrane proteins that arespecific for other diseases. Diseased cells can be isolated and used togenerate lipoparticles and antibodies as described herein. Theantibodies can then be used as diagnostic tools to identify diseasedcells and in some embodiments, the antibodies can be used to treat thedisease if the antibodies are found to kill the diseased cells.

Antibodies can also be generated against membrane proteins usinglipoparticles, viruses, or virus-like particles, and a phage libraryand/or ribosome display (see, for example Hoogenboom H R. “Overview ofantibody phage-display technology and its applications.” Methods MolBio. (2002); 178:1-37; He, M. et al., Brief Funct Genomic Proteomic.(2002) July; 1(2):204-12; D, Pluckthun A, et al., J Immunol Methods.(2004) July; 290(1-2):51-67). In some embodiments, the librariescomprise a library of monoclonal antibody binding domains.

The particulate nature of lipoparticles makes them comparable tokilled-virus vaccines currently used to successfully elicit immuneresponses (e.g. humoral and cellular). The ability to place non-viralmolecules within such an immunogen allows lipoparticles to have directapplication to both preventative and therapeutic vaccines.

Thus, the present invention provides methods of eliciting immuneresponses against membrane proteins. The lipoparticles can be introducedinto an animal to elicit an immune response. Examples of animals orsubjects from which immune responses can be elicited from include, forexample, mice, rats, chickens, sheep, horses, goats, pigs, non-humanprimates, or humans.

In some embodiments, the use of lipoparticles as an immunogen willelicit a cytotoxic T-cell response as well as a humoral (antibody)response. Therefore, lipoparticles can be used in the preparation ofvaccines to protect or treat a disease, disorder, or condition. Theantibodies generated in response to using a lipoparticle as an immunogencan be used, for example, as therapeutics or as diagnostics (e.g.identification of proteins, disease states, and the like). The immuneresponses generated can treat or protect against, without limitation,cancer (e.g. breast cancer, prostate cancer, lung cancer, blood-bornecancer (e.g. multiple myeloma), bone cancer, leukemias, head and neckcancers, brain cancer, and the like), viral and bacterial infectiousagents (e.g. HIV, HSV, HCV, Hepatitis A, Hepatitis B, coronaries, SARS,West Nile Virus, anthrax, vaccinia, and the like).

The present invention also provides antigenic compositions comprisinglipoparticles with a cellular protein of interest (e.g. an exogenousprotein or a naturally expressed protein) and a pharmaceuticalacceptable carrier.

The immunogen can be prepared in dose form by well-known procedures. Theimmunogen can be administered, for example, parenterally (e.g.intravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, (e.g., by inhalation or insufflation, or intrathecal orintraventricular administration), topically (e.g. ophthalmic, vaginal,rectal, intranasal, transdermal), orally, intramuscularly,subcutaneously, pulmonary administration, or intranasally. Forparenteral administration, such as intramuscular injection, theimmunogen may be combined with a suitable carrier, for example, it maybe administered in water, saline, or buffered vehicles with or withoutvarious adjuvants or immunostimulating agents such as aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate, berylliumsulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-wateremulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum, Bordetella pertussis, polyribonucleotides,sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes,levamisole, DEAE-dextran, blocked copolymers or other syntheticadjuvants. Such adjuvants are available commercially from varioussources, for example, Merck Adjuvant 65 (Merck and Company, Inc.,Rahway, N.J.). Compositions for parenteral, intravenous, intrathecal orintraventricular administration may include sterile aqueous solutionswhich can also contain buffers, diluents and other suitable additivesand are preferably sterile and pyrogen free.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers; aqueous, powder oroily base; thickeners and the like can be used. Compositions for oraladministration include powders or granules, suspensions or solutions inwater or non-aqueous media, capsules, sachets or tablets. Thickeners,flavoring agents, diluents, emulsifiers, dispersing aids or binders maybe desirable.

The proportion of immunogen and immunostimulating agent can be variedover a broad range so long as both are present in effective amounts. Forexample, aluminum hydroxide can be present in an amount of about 0.5%w/v of the vaccine mixture. On a per dose basis, the concentration ofthe immunogen can range from about 0.015 μg to about 1.5 mg per kilogramper body weight. A preferable dosage range is from about 1.5 μg/kg toabout 0.043 mg/kg of body weight. A suitable dose size in humans isabout 0.1-1 ml, preferably about 0.1 ml. Accordingly, a dose forintramuscular injection in humans, for example, would comprise 0.1 mlcontaining 1.5 μg/kg immunogen in admixture with 0.5% aluminumhydroxide.

The dosage administered can also vary and depend upon factors such as:pharmacodynamic characteristics; mode and route of administration; age,health, and weight of the recipient; nature and extent of symptoms; kindof concurrent treatment; and frequency of treatment. Usually, the dosageof an immunogenic composition can be about 1 to 3000 milligrams per 50kilograms of body weight; preferably 10 to 1000 milligrams per 50kilograms of body weight; more preferably 25 to 800 milligrams per 50kilograms of body weight. In some embodiments, 8 to 800 milligramsadministered to an individual per day in divided doses 1 to 6 times aday, or in sustained release form, is effective to obtain desiredresults. Formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.

The compositions according to the present invention can be administeredas a single dose or in multiple doses. The compositions of the presentinvention can be administered either as individual therapeutic agents orin combination with other therapeutic agents. The treatments of thepresent invention may be combined with conventional therapies, which maybe administered sequentially or simultaneously.

In the instance of vaccines, the vaccine can also be combined with othervaccines for other diseases to produce multivalent vaccines. It can alsobe combined with other medicaments such as antibiotics.

The present invention also provides pharmaceutical compositions thatcomprise the immunogens of the invention and pharmaceutically acceptablecarriers or diluents. The compositions of the present invention may beformulated by one having ordinary skill in the art with compositionsselected depending upon the chosen mode of administration. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text. In carrying out methods ofthe present invention, immunogenic and/or antigenic compositions of thepresent invention can be used alone or in combination with otherdiagnostic, therapeutic or additional agents. Such additional agentsinclude excipients such as flavoring, coloring, stabilizing agents,thickening materials, osmotic agents and antibacterial agents. Suchagents may enhance the lipoparticle use in vitro or in vivo, thestability of the composition during storage, or other propertiesimportant to achieving optimal effectiveness.

For parenteral administration, the immunogenic and/or antigeniccompositions of the invention can be, for example, formulated as asolution, suspension, emulsion or lyophilized powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes and nonaqueous vehicles such as fixedoils can also be used. The vehicle or lyophilized powder may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

The compositions of the present invention may be administered by anymeans that enables the active agent to reach the site of action. Becauselipoparticles may be subject to being digested when administered orally,parenteral administration, i.e., intravenous, subcutaneous, transdermal,intramuscular, can be used to optimize absorption. Intravenousadministration may be accomplished with the aid of an infusion pump. Thecompositions of the present invention can be formulated as an emulsion.Alternatively, they can be formulated as aerosol medicaments forintranasal or inhalation administration. In some cases, topicaladministration can be desirable.

Depending upon the disease or disorder to be treated, the compositionsof the present invention may be formulated and administered to mosteffectively to treat the disease or disorder. Modes of administrationwill be apparent to one skilled in the art in view of the presentdisclosure.

Incorporation of membrane-bound receptors and/or cellular proteins intolipoparticles avoids the complex issues associated with membrane proteinsolubilization, purification, and reconstitution. Once produced,lipoparticles are easy to purify, in addition to being quite stable.Thus, the use of lipoparticles allows the cell and/or viral machinery todo the work of reconstituting the protein of interest into a native,biological membrane. Importantly, the lipid membrane surrounding thelipoparticle is not significantly different from the membrane thatsurrounds the cell from which the lipoparticle was derived, thus theincorporated proteins are presented in a native lipid environment.

Advantages of using lipoparticles as compared to a commonly usedalternative technique in which vesicles are prepared from membranes ofcells expressing the desired receptor are listed in Table 8. Briefly,antigens prepared from cell membranes are heterogeneous, receptors maybe misoriented, the antigens are impure, and are not concentrated.Vesicles containing antigens derived from cells also tend to beheterogeneous in size, not particularly stable, and can be contaminatedwith undesirable proteins. Table 8 compares the advantages of usinglipoparticles to produce antigens over other methods (e.g. purifyingrecombinant proteins, peptide synthesis, transient cellular expression,and stable cell lines).

TABLE 8 Advantages of producing antigens using lipoparticles versusconventional methodologies for Mab development. Traditional Methods ofProducing Antigens Technologies and Advantages Transient Stable forAntibody Development Cellular Cell to Complex Proteins LipoparticlesExpression Lines Proteins Peptides Stable Receptor Expression X X XRetention of Receptor Structure X X X Application to Toxic Proteins X XX X Use of Transient Protein Expression X X High Protein Expression X XX X X Protein Concentration X X X Rapid Purification X X X EnrichedProteins X X X Intracellular Receptors X X X Particulate Size (~100 nM)X

The present invention also provides for methods for treating a diseaseusing the antibodies produced using the methods of the presentinvention. The antibodies can be used to treat, for example, cancer(e.g. breast cancer, cervical, lung cancer, ovarian cancer, bone cancer,leukemia, myelomas, melanomas, skin cancer, head and neck cancer,bladder cancer, liver cancer, pancreatic cancer, esophageal cancer,colon cancer, and the like), rheumatoid arthritis, osteo-arthritis,crohn's disease, viral infections (e.g. HIV, Hepatitis A, B, and C,Human Papilloma virus, West Nile Virus, SARS, Ebola, smallpox,influenza, common cold, viral meningitis, Epstein-Barr virus, herpessimplex virus, and the like), neurological diseases (e.g. Alzheimer'sdiseases, Parkinson's disease, and the like) multiple sclerosis,bacterial infections (e.g. salmonella, E. coli, anthrax, bacterialmeningitis, botulism, Bordetella pertussis, Streptococcus), and thelike. The antibodies can be administered as compositions as describedfor other compositions described herein.

Lipoparticles containing a Gag fusion protein can also be used asimmunogens to elicit antibodies against the fusion protein.Lipoparticles will be produced in large quantities and will containlarge amounts of the fusion protein in every lipoparticle, linked to theGag structural protein. When injected into mice, the fusion protein canserve as a source of antigen for immune presentation.

The present invention provides for kits for eliciting an immune responseagainst a membrane protein, wherein the kits comprises a lipoparticlecomprising a cellular protein of interest and a protocol forimmunization and/or instructional material.

While traditional protein transfection localizes exogenous protein intothe target cell's cytoplasm or nucleus, membrane protein transfectionlocalizes membrane proteins onto a target cell's plasma membrane. Thepresent invention provides methods of transferring a membrane proteinfrom the bilayer of a lipoparticle to a target cell's membrane bilayer.Transfection can be facilitated by fusion and/or endocytosis of alipoparticle, which results in the transfer of the lipoparticle'smembrane proteins to the target cell.

Membrane protein transfection extends the benefits of proteintransfection to membrane proteins. In addition to allowing transfectionof proteins inherently toxic to cells, membrane protein transfectiontechniques can be used on cells difficult to transfect with DNA.Additional obstacles that can be overcome using membrane proteintransfection include, for example, proteins that are otherwise difficultto express on the plasma membrane, proteins that have difficultytrafficking to the plasma membrane, and proteins that do not foldcorrectly. Additionally, because the lipoparticle already contains theintact protein, post-translational and splicing modifications can bespecifically controlled using lipoparticles for membrane proteintransfection to specify the protein product that is contained within acell.

Fusing lipid membranes is known to one of ordinary skill in the art.Therefore, any technique that can be used to fuse one lipid membranewith another lipid membrane can be used for membrane proteintransfection using lipoparticles. Examples of methods and reagents thatcan be used to fuse two lipid bilayers include, but are not limited to,exposure to low pH, exposure to high calcium concentrations, calciumphosphate precipitate, PEG8000, DNA transfection reagents (e.g.lipofectamine and effectene), Peptide Transduction Domains (e.g.Chariot™, protein delivery reagent), sonication, viral fusion proteinssuch as VSV Envelope, detergents (e.g. .beta.-octylglucoside), alkanes,exposure to DEAE Dextran, centrifugation, and electroporation.Additional methods of lipid fusion and/or membrane permeabilization areindicated in the table below.

Method of Plasma Membrane Breach Chemical ATP Influx pinocyticcell-loading reagent (I-14402) EDTA Ca3(PO4)2 DEAE-dextran Alpha Toxinof Staphylococcus aureus Transferrin polylysine Vehicle Red blood cellfusion Vesicle and liposome fusion Mechanical Microinjection Hypoosmoticshock Osmotic lysis of pinosomes Scrape loading Agitation in coldSonication (mild) High-velocity microprojectiles Glass beads Scratchingto wound culture Electrical ElectroporationTable 9 reproduced from Molecular Probes catalog. Table adapted fromMcNeil, P. L., in Fluorescence Microscopy of Living Cells in Culture,Part A.

To detect whether protein transfection has occurred using lipoparticlesany method can be used. Examples of detection methods include, but arenot limited to, calcium flux to detect GPCRs transfected into the cell,ion channel conductance to detect ion channels transfected into thecell, membrane fusion, lipid dye mixing, fluorescence quenching, andWestern blot.

For example, lipid dye mixing can be used to monitor the fusion of thetwo lipid membranes. A lipoparticle comprising a membrane protein can begenerated that incorporates a lipid dye. When the lipoparticle fuseswith its target (e.g. another cell, viral particle, bacteria,lipoparticle, and the like) the dye will also enter and mix with thetarget. A change in the dye's fluorescence or intensity can be used tomonitor the progress and completion of the protein being transfectedinto the target.

Western blot can also be used to monitor protein transfection. Afterfusing the lipoparticle with its target cell, the protein can bedetected in the target by isolating the target cell (e.g. washing awayunincorporated molecules) and performing a Western blot to detect thetransfected protein. Similar to Western blot, immunofluorescence canalso be used. The target can be contacted with either a labeled primaryantibody that recognizes the transfected protein or through the use of alabeled secondary antibody. The labels can be any label that isdetectable and can include, for example, FITC, Texas-Red, and the like.A radioactive label can also be used.

The present invention provides methods of transfecting a protein into acell comprising contacting the cell with a lipoparticle comprising theprotein. In some embodiments the lipoparticle comprises a viral proteincomponent. In some embodiments the viral protein component comprises aviral structural protein. An example of a viral structural proteinincludes, but is not limited to, Gag. In some embodiments, thelipoparticle is integration incompetent, protease incompetent, reversetranscription incompetent, or combinations thereof.

Any protein can be transfected from a lipoparticle to a cell. Theprotein can be, for example, a membrane protein. Membrane proteinscomprise a diverse group of proteins including, for example, G-proteincoupled receptors, ion channels, receptors, tyrosine kinase receptors,and the like. In some embodiments, the membrane protein is the cysticfibrosis transmembrane regulator (CFTR).

The present invention also provides for methods for the transfer of aprotein from a lipoparticle to a cell to correct or modulate a defect inthe cell. In some embodiments, the protein modulates at least oneproperty of the cell. The property of the cell can be, for example, butis not limited to, growth property, ion-conductance property, signalingproperty.

The present invention also provides for methods for the transfer of aprotein from a lipoparticle to a cell to enhance the function of a cell.In some embodiments, the protein is not expressed within the targetcell, or is expressed at low levels, and protein transfection of themembrane protein into the target cell leads to an enhancement of theproperties of that cell. The property of the cell can be, for example,but is not limited to, growth property, ion-conductance property,signaling property.

Although, the protein that is transfected can be wild-type, the proteincan also be modified. In some embodiments, the modification is amutation, deletion, insertion, post-translational modification, chimericmodification, or combinations thereof.

The cell that the protein is transfected into can be any type of cell.Examples of cells that a protein can be transfected into include, butare not limited to, primary cells, stem cells, cancer cells, quiescentcells, terminally differentiated cells, and the like. In someembodiments, examples of cells also include, but are not limited to,293T, 293, HeLa, Vero, BHK, CHO, NT-2, 3T3, QT6 cells, and the like. Insome embodiments, the type of cell is a cell that cannot be transfectedwell with a nucleotide molecule (e.g. plasmid). As used herein, the term“cannot be transfected well” refers to a cell that when transfected witha nucleotide molecule does not provide an effective amount of a protein.An “effective amount of a protein” as used herein, refers to an amountthat is determined to be necessary to perform a function in the cell.The function can be any function or use of the protein. For example, thefunction can be the ability to measure the expression of the protein orthe function can be the ability of the protein to modulate a property ofthe cell. Other functions are also included within the scope of thepresent invention. As used herein, the term “cannot be transfected well”also refers to a low percentage of cells being transfected with anucleotide molecule. As used herein, the term “low percentage of cells”refers to less than less than 50%, less than 40%, less than 30%, lessthan 20%, less than 10%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% of cells being transfected with a nucleotidemolecule. In some embodiments, the cell is taken from a subject and thentransfected with a lipoparticle comprising a protein. In someembodiments, this is referred to as “ex vivo” transfection. Methods ofisolating cells from a subject or individual are well known to those ofskill in the art.

The present invention provides cells comprising a membrane protein thatwas transfected into the cell using lipoparticles of the presentinvention. In some embodiments, the cell comprises at least a portion ofa lipoparticle's membrane.

The present invention also provides methods of treating a diseasecomprising administering a lipoparticle comprising a membrane protein.Examples of diseases include, but are not limited to cystic fibrosis andcancer. In some embodiments the lipoparticles are administered to ananimal. In some embodiments, the animal is a human, a mouse, a rat, adog, a cat, a horse, and the like.

The present invention also provides methods for administeringlipoparticles comprising a protein and a pharmaceutically acceptablecarrier. In some embodiments, the protein is a membrane protein.

The lipoparticle can be prepared in dose form by well-known procedures.The lipoparticle can be administered, for example, parenterally (e.g.intravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, (e.g., by inhalation or insufflation, or intrathecal orintraventricular administration), topically (e.g. ophthalmic, vaginal,rectal, intranasal, transdermal), orally, intramuscularly,subcutaneously, pulmonary administration, or intranasally. Forparenteral administration, such as intramuscular injection, thelipoparticle may be combined with a suitable carrier, for example, itmay be administered in water, saline, or buffered vehicles with orwithout various adjuvants or immunostimulating agents such as aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate, berylliumsulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-wateremulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum, Bordetella pertussis, polyribonucleotides,sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes,levamisole, DEAE-dextran, blocked copolymers or other syntheticadjuvants. Such adjuvants are available commercially from varioussources, for example, Merck Adjuvant 65 (Merck and Company, Inc.,Rahway, N.J.). Compositions for parenteral, intravenous, intrathecal orintraventricular administration may include sterile aqueous solutionswhich can also contain buffers, diluents and other suitable additivesand are preferably sterile and pyrogen free.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers; aqueous, powder oroily base; thickeners and the like can be used. Compositions for oraladministration include powders or granules, suspensions or solutions inwater or non-aqueous media, capsules, sachets or tablets. Thickeners,flavoring agents, diluents, emulsifiers, dispersing aids or binders maybe desirable.

On a per dose basis, the concentration of the lipoparticle can rangefrom about 0.015 μg to about 1.5 mg per kilogram per body weight. Apreferable dosage range is from about 1.5 μg/kg to about 0.043 mg/kg ofbody weight. A suitable dose size in humans is about 0.1-1 ml, or about0.1 ml. Accordingly, a dose for intramuscular injection in humans, forexample, would comprise 0.1 ml containing 1.5 μg/kg lipoparticle inadmixture with 0.5% aluminum hydroxide. The dose of a lipoparticle canalso be administered based on the number of lipoparticles per kilogramof body weight. In some embodiments, the dose is about 5lipoparticles/kg, about 100 lipoparticles/kg, about 1000lipoparticles/kg, about 10,000 lipoparticles/kg, about 100,000 orlipoparticles/kg, about 500,000 lipoparticles/kg, about 1×10⁶lipoparticles/kg, about 1×10⁷ lipoparticles/kg, about 1×10⁸lipoparticles/kg, about 1×10⁹ lipoparticles/kg, about 1×10¹⁰lipoparticles/kg, about 1×10¹¹ lipoparticles/kg, or about 1×10¹² or morelipoparticles/kg. In some embodiments more than 500,000 lipoparticles/kgare administered as a dose.

The dosage administered can also vary and depend upon factors such as:pharmacodynamic characteristics; mode and route of administration; age,health, and weight of the recipient; nature and extent of symptoms; kindof concurrent treatment; and frequency of treatment. Usually, the dosageof a lipoparticle composition can be about 1 to 3000 milligrams per 50kilograms of body weight; preferably 10 to 1000 milligrams per 50kilograms of body weight; more preferably 25 to 800 milligrams per 50kilograms of body weight. In some embodiments, 8 to 800 milligramsadministered to an individual per day in divided doses 1 to 6 times aday, or in sustained release form, is effective to obtain desiredresults. Formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.

The compositions according to the present invention can be administeredas a single dose or in multiple doses. The compositions of the presentinvention can be administered either as individual therapeutic agents orin combination with other therapeutic agents. The treatments of thepresent invention may be combined with conventional therapies, which maybe administered sequentially or simultaneously.

For parenteral administration, the lipoparticle compositions of theinvention can be, for example, formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 5% human serumalbumin. Liposomes and nonaqueous vehicles such as fixed oils can alsobe used. The vehicle or lyophilized powder may contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques. For example, a parenteralcomposition suitable for administration by injection is prepared bydissolving 1.5% by weight of active ingredient in 0.9% sodium chloridesolution.

The compositions of the present invention may be administered by anymeans that enables the active agent to reach the site of action. Becauselipoparticles may be subject to being digested when administered orally,parenteral administration, i.e., intravenous, subcutaneous, transdermal,intramuscular, can be used to optimize absorption. Intravenousadministration may be accomplished with the aid of an infusion pump. Thecompositions of the present invention can be formulated as an emulsion.Alternatively, they can be formulated as aerosol medicaments forintranasal or inhalation administration. In some cases, topicaladministration can be desirable.

Depending upon the disease or disorder to be treated, the compositionsof the present invention may be formulated and administered to mosteffectively to treat the disease or disorder. Modes of administrationwill be apparent to one skilled in the art in view of the presentdisclosure.

Measurement of a Virus, Virus Like Particles, Lipoparticles and OtherUses

The ability to produce lipoparticles does not confer the ability todetect, visualize, count, or measure the lipoparticles. The presentinvention provides these abilities. In addition, the membrane proteinswithin the lipoparticles can be quantified, their density within eachlipoparticle measured, and the purity of the lipoparticles determined.The methods disclosed herein are described in relation to lipoparticles,but can also be applied to naturally occurring or laboratory strains ofviruses, viral particles, or virus-like-particles. Therefore, as usedherein, the term “particle” refers to any particle comprising aphospholipid layer comprising a viral core protein and an additionalprotein, a virus, viral particle, virus-like-particle, or lipoparticle.In some embodiments, the additional protein is a membrane protein.

The present invention provides for particles comprising a fluorophore.In some embodiments the particle is a virus, a viral particle,virus-like-particle, or a lipoparticle. The fluorophore can be anycompound or composition that fluoresces. In some embodiments, thefluorophore is a fluorescent protein or a fluorescent dye. Thefluorescent protein can be a fusion protein that comprises a fluorescentprotein and a non-fluorescent protein. Examples of fluorescent proteinsinclude, but are not limited to, Green Fluorescent Protein (GFP), YellowFluorescent Protein (YFP), Blue Fluorescent Protein (BFP), CyanFluorescent Protein (CFP), DsRED, AsRED, AmCyan, HcRed, ZsGreen,ZsYellow, or variants thereof. In some embodiments, the fluorescentprotein comprises a viral structural protein (e.g. Gag). The use of acomposition comprising a lipoparticle and a fluorescent protein allowsfor the lipoparticles to be detected and/or quantified in ways that havenot been done previously. Making lipoparticles with specific viralstructural proteins and/or specific membrane proteins is described inU.S. Patent Application US 2002/0183247A1, U.S. Application Ser. No.60/491,477, U.S. Application Ser. No. 60/491,633, U.S. Application Ser.No. 60/498,755, and U.S. Application Ser. No. 60/502,478. Additionally,membrane proteins can be transfected into other cells or particles asdescribed herein and in U.S. Provisional Application 60/509,677, filedOct. 7, 2003.

Fluorophores can also be incorporated into particles using fluorescentdyes. In some embodiments the fluorescent dye is a hydrophobic dye. Ahydrophobic fluorescent dye is a dye that fluoresces more strongly in anon-aqueous environment. In some embodiments, the hydrophobicfluorescent dye does not appreciably fluoresce in an aqueousenvironment. In some embodiments, the hydrophobic fluorescent dye doesnot fluoresce in an aqueous environment. As used herein, the term “doesnot appreciably fluoresce in an aqueous environment” refers to thefluorescent property of a compound (e.g. dye). In some embodiments thefluorescence of a compound that does not appreciably fluoresce in anaqueous environment is about 10% less, about 20% less, about 30% less,about 40% less, about 50% less, about 60% less, about 70% less, about80% less, about 90% less, about 91% less, about 92% less, about 93%less, about 94% less, about 95% less, about 96% less, about 97% less,about 98% less, about 99% less, or 100% less in an aqueous environmentthan it fluoresces in a non-aqueous environment. Examples of non-aqueousenvironments include, but are not limited, a lipid bilayer, cellmembrane, and the like. Fluorescent dyes can be incorporated intoparticles as described in U.S. Application Ser. No. 60/498,755. In someembodiments, dyes that fluoresce strongly in both aqueous and lipidenvironments, the dye can be separated from the lipoparticles prior tovisualization, for example, by a gel filtration spin column.

Examples of dyes that bind and/or label lipids include, but are notlimited to, Amphiphilic dyes; DiA; 4-Di-10-ASP; FASTDiA; FM 1-43; FM4-64; FM 5-95; NBD; TMA-DPH; TMAP-DPH; ANS; MBDS; BADS;4-amino-4′-benzamidostilbene-2,2′-disulfonic acid, disodium salt (MBDS);1-anilinonaphthalene-8-sulfonic acid (1,8-ANS);2-anilinonaphthalene-6-sulfonic acid (2,6-ANS); bis-ANS(4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt),(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene(BODIPY® 665/676); 1,10-bis-(1-pyrene)decane;1,3-bis-(1-pyrenyl)propane; Dapoxyl® sulfonic acid sodium salt;4-(dicyanovinyl)julolidine (DCVJ);6,8-difluoro-4-heptadecyl-7-hydroxycoumarin (C17DiFU);4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene(BODIPY® 493/503);4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY®505/515) 4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole (NBDdihexadecylamine);4-(N,N-dimethyl-N-tetradecylammonium)methyl-(7-hydroxycoumarin) chloride(U-6); 1,6-diphenyl-1,3,5-hexatriene (DPH);5-dodecanoylaminofluorescein; 6-dodecanoyl-2-dimethylaminonaphthalene(laurdan); fluorescein octadecyl ester; 4-heptadecyl-7-hydroxycoumarin;5-hexadecanoylaminofluorescein;6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalenechloride (patman); Nile red; Di-4-ANEPPS; Di-8-ANEPPS;5-octadecanoylaminofluorescein;N-octadecyl-N′-(5-(fluoresceinyl))thiourea (F18); octadecyl rhodamine Bchloride (R18); 3-(4-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid(DPH propionic acid);N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammoniump-toluenesulfonate (TMAP-DPH); N-phenyl-1-naphthylamine;6-propionyl-2-dimethylaminonaphthalene (prodan); 1-pyrenebutanol;1-pyrenenonanol; 1-pyrenesulfonic acid, sodium salt;2-(p-toluidinyl)naphthalene-6-sulfonic acid, sodium salt (2,6-TNS);1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatrienep-toluenesulfonate (TMA-DPH); diI; diS; diO; Oxonol VI; JC-1; DiSC3(5);DiSC3; CC2-DMPE; DiSBAC2(3); DiSBAC4(3); VABSC-1; Rhodamine 421; and thelike.

The present invention also provides methods for detecting othersub-microscopic particles. Previously, it has been very difficult, timeconsuming, and expensive to detect and/or visualize such particles.Techniques that are expensive and laborious, such as electron microscopy(EM), have been previously used to detect and quantify particles.However, the present invention provides methods that are easier to use,less expensive, and less laborious. By using a composition comprising aparticle and a fluorophore one can now easily detect and quantify theparticles with methods that were thought to have not been practical ortechnically feasible.

In some embodiments the detection of a particle is done on the basis ofthe particle comprising a fluorophore (e.g. fluorescent dye orfluorescent protein). The detection can be done using any means that canmeasure fluorescence including, but not limited to, microscopy, flowcytometry, immunofluorescence, and the like. In some embodiments, themicroscopy is performed with a microscope containing an epifluorescentlight. In some embodiments, the microscopy is performed with a confocalmicroscope. In some embodiments, if the fluorescent is bright enough, itis contemplated that a fluorescent microscope may not be necessary todetect the fluorophore in the particles.

Being able to detect particles can allow the particles to be quantifiedor counted. The counting can be done by any means including, forexample, using a hemocytometer and a microscope, flow cytometry, dynamiclight scattering, static light scattering, quantitative lightscattering, immunofluorescence, reflectance, absorbance, and the like.Being able to count particles can enable doses to be more easily andaccurately calculated when administering a composition that containsparticles to an animal or an individual. Counting particles can also beused to determine the concentration and/or purity of particles in asample. Thus, the present invention provides methods for determining theconcentration and/or purity of particles in a sample.

In some cases, the results of quantification are not returned in termsof absolute number of particles. For example, the particles can becounted by dynamic light scattering, static light scattering,quantitative light scattering, or reactivity with a fluorescentmolecule, and the results from these assays are in units of light,intensity of light, or a similar measurement unit, not particle numbers.In some embodiments, the particles can be quantified by quantitative PCRor quantitative RT-PCR, by detection of Gag by Western blot or dot blot,or by detection of membrane phospholipids or cholesterol, resultingagain in relative units of detection. The units of detection, however,can be readily converted to absolute particle numbers by correlating theresults of such an assay with a standard curve using known quantities oflipoparticles, beads, DNA, Gag protein, or other standards of knownquantity.

The purity of a lipoparticle preparation can be calculated bydetermining the number of particles in a sample and dividing that numberby the total protein concentration of the sample. Determining theprotein concentration of a sample is well known to those of skill in theart and can be done using any method. Some commercially availableproducts that can be used include, but are not limited to, BCA assay kit(Pierce, Rockford, Ill.) and microBCA assay kit (Pierce, Rockford,Ill.), NanoOrange (Molecular Probes, Eugene, Oreg.). The proteinconcentration can be calculated in any units including, for example,ug/μl, ug/ml, mg/μl, mg/ml, mg/l, and the like. Therefore, in someembodiments the equationPurity of Particles=(Number of Particles per unit volume)/(Total Proteinper unit volume)can be used to determine the number of particles per unit weight (e.g.μg, mg, and the like). This can allow one to calibrate experiments andcompositions more accurately as compared to other methods known in theart. In some embodiments the purity is determined by taking the numbergenerated by the equationPurity of Particles=(Number of Particles per unit volume)/(Total Proteinper unit volume) anddividing the purity of particles number by the theoretical weight of thelipoparticle or the theorectical protein weight of the particle. If thiscalculation is about 1 or equal to 1, the particles are said to be pure.If the number is greater than one, the particles are less pure. In someembodiments, if the number is greater than 1.1, greater than 1.2,greater than 1.3, greater, than 1.4, greater than 1.5, greater than 1.6,greater than 2.0 the particles are not pure.

In some embodiments, the quantifying the number of said membrane proteincomprises purifying the membrane protein from the lipoparticles anddetermining the concentration of protein in the purified membraneprotein sample. In some embodiments, the quantifying the number of themembrane protein comprises determining the number of ligand bindingsites per mg protein in the sample. The quantifying the number can bedone with a method comprises a Western blot, dot blot, or total proteinstain SDS-PAGE. In some embodiments, a total protein stain SDS-PAGEcomprises a Coomassie, Sypro, or silver stain.

In some embodiments, the total protein stain SDS-PAGE comprisescalculating the percent of total protein represented by the membraneprotein. In some embodiments, the Western blot, dot blot, or totalprotein stain SDS-PAGE comprises a protein standard.

As discussed above, when detecting and/or counting lipoparticles one canuse flow cytometry. To overcome limitations in the resolution of flowcytometry, in some embodiments, the present invention providescompositions comprising particles and beads. In some embodiments, theparticles are attached to the beads either through non-covalent orcovalent interactions. The attachment can be through, for example, abiotin-avidin interaction, wheat germ agglutinin interaction, or apoly-lysine interaction, where the bead has one molecule or compoundthat interacts with a molecule or compound present on the particle. Thenumber of particles that can be attached to a bead can be varied basedon the conditions that the particles and the beads are contacted withone another. In some embodiments, the number of particles attached toeach bead is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more. The conditions that can be varied includetemperature, concentration of lipoparticles, concentration of the beads,and the like. The beads can be any type of bead made out of any materialthat is suitable for flow cytometry. The size of the beads can also beany size. In some embodiments the diameter of the bead is about 1 um,about 2 um, about 5 um, about 10 um, about 15 um, about 20 um, about 30um, about 40 um, or more than 41 um. Once the particles are attached tobeads, they can be counted using the flow cytometer. In someembodiments, the bead comprises a fluorophore or the particle comprisesa fluorophore to enable detection by the flow cytometer.

Being able to count the number of particles can also allow one todetermine the number of membrane proteins or a specific membrane proteinthat is present on the particle. A person of ordinary skill in the artcan calculate the amount of a membrane protein within a sample usingstandard techniques known including, but are not limited to, WesternBlot, ELISA, and the like. When these techniques are used in conjunctionwith a standard (as described herein) the amount of a protein can becalculated in a sample. Then using the equationNo. of Membrane Proteins per particle=(No. of Membrane Protein per μl ofsample)/(No. of lipoparticles per μl of sample)

By using this equation one can determine the amount of a specificmembrane protein per particle. This can be used to standardize doses ofa composition, as well as monitor how well or how much of a membraneprotein is incorporated into a particle during the production of theparticle. In some embodiments, it is determined that there are at least1, at least 5, at least 10, at least 20, at least 30, at least 40, atleast 50, at least 100, at least 200, at least 1000, at least 10,000, ormore specific membrane protein molecules per particle. In someembodiments there are about 1 to about 10,000, about 1 to about 1,000,about 1 to about 500, about 1 to about 200, about 1 to about 100, about1 to about 50, about 5 to about 500 specific membrane protein moleculesper particle. As used herein, the term “specific membrane protein”refers to a membrane protein that is being quantified. This does notrefer to the total number of membrane proteins on or in the surface of aparticle.

The present invention provides methods for determining the density of amembrane protein on the surface of a particle. The size of a particlecan be determined using dynamic light scattering. Based on the size ofthe particle one can calculate the density of the membrane protein usingthe equationSurface Density of a Membrane Protein=(No. of Membrane Proteins perparticle)/(Size per particle)

The equation will result in an answer with units of number of membraneparticles per unit size which equals the surface density or probabledistribution of the membrane protein on or in the particle.

Based on the ability to detect particles using fluorophores, the presentinvention further provides methods of detecting the binding of compoundsto particles. In some embodiments the methods detect binding ofcompounds to lipoparticles. The compound can be any compound that isthought to bind to, or it is wanted to know if it binds to, theparticles including, but not limited to, ligands, peptides, proteins,antibodies, organic chemical compounds (e.g. small molecular weightcompounds), or inorganic chemical compounds. In some embodiments, thecompound comprises a fluorophore or fluorescent label.

The compounds can be contacted with the particle to determine if thecompound can bind to the particle. To determine if the compound can bindto the lipoparticle one can detect the binding through the fluorescenceof bound compound when the compound comprises a fluorophore orfluorescent label. Detection methods include, but are not limited to,flow cytometry, immunofluorescence, sensors, microscopy, and the like.Sensors and using them with particles is described, for example, in U.S.Application No. 60/491,633. In some embodiments, the compound isimmobilized on the sensor surface. In some embodiments, the particle isimmobilized on the sensor surface.

The present invention also provides methods of identifying compoundsthat bind to the same site as that of a compound that is known to bindto a particle or to a site that prevents the compound known to bind to aparticle by some other mechanism such as a steric hindrance orallosteric change in the particle. This can be detected using theability to detect particles using the present invention. The methodcomprises contacting a first compound with a particle to which a secondcompound is already bound to or to which it is known that a secondcompound can bind to, but has not yet been contacted with the particle.In some embodiments, the first compound comprises a fluorophore. In someembodiments, the second compound comprises a fluorophore. In someembodiments, the first and second compounds comprise differentfluorophores. One can detect if the second compound is prevented frombinding to the particle by the change in fluorescence that would beobserved. If only the second compound comprises a fluorophore, then adecrease in fluorescence once the first compound is brought in contactwith the particle would indicate that the first compound prevents orinhibits the second compound from binding to the particle. If only thefirst compound comprises a fluorophore, then an increase in fluorescencewould indicate that the second compound is inhibited or prevented frombinding to the particle by the presence of the first particle. In someembodiments, the particle comprises a fluorophore and the fluorescencecan indicate whether or not a compound is bound to the particle. In someembodiments, when the second compound is bound to the particle, theparticle fluoresces, whereas when the first compound binds to theparticle the particle does not fluoresce and vice versa. Therefore, thechanges in fluorescence can be used to determine if the first compoundinhibits or prevents the second compound from binding to the particle.The changes in fluorescence, either associated with the particle or thecompound, can also be used to determine if the first compound can bindto the particle. Methods of measuring fluorescence are known to those ofordinary skill in the art.

The present invention also provides methods for detecting the structuralintegrity of a membrane protein on or in a particle. In someembodiments, the method comprises contacting the particle with anantibody that binds to the membrane protein and detecting the binding ofthe antibody to the particle. In some embodiments, the detection of theantibody being bound to the particle indicates that the structuralintegrity of the membrane protein is intact. “Structural integrity” asused herein refers to the proper folding and presentation of a membraneprotein on or in the surface of a particle. In some embodiments itrefers to the proper folding and expression on or in the surface of alipoparticle.

The binding of an antibody to a membrane protein can indicate structuralintegrity because in some embodiments, the antibody is aconformationally dependent antibody. A “conformationally dependentantibody” refers to an antibody that only binds to a protein or membraneprotein when the protein is properly folded and retains its correctstructure. In some embodiments the antibody can recognize aconformationally active protein (e.g. activated receptor) or aconformationally inactive protein. Any method can be used to determinestructural integrity, including Virus-Detection Elisa,Antibody-Detection Viral Elisa, a sensor, flow cytometry, andimmunofluorescence staining. In some embodiments, centrifugation can beused to isolate the particles before or during the determination of thestructural integrity process. In some embodiments, methods comprisingVirus-Detection Elisa, Antibody-Detection Viral Elisa, orimmunofluorescence further comprise a centrifugation step or spin downstep. Centrifugation can be used in any method to facilitate thedetection of the particles.

Detection of Antigens and/or Ligands Using a Lipoparticle

Lipoparticles can also be used here to construct pathogen, antibody, andligand biosensors that utilize cellular single-transmembrane signalingmachinery. By incorporating the necessary target-recognition elements (asingle-transmembrane protein capable of independent or antibody-assistedbinding of pathogens, ligands and antibodies) and reporter elements(e.g. fluorescent and/or luminescent proteins), one can use an assay forthe detection of pathogen antigens, pathogen-specific antibodies, andreceptor ligands. Uses of these assays include, but are not limited to,evaluating biological, food and environmental samples for the presenceof infectious agents and contaminants for a range of purposes includingbioterrorism screening, disease diagnosis, epidemiological surveys, foodcontamination assessment, and the like. The lipoparticles can also beused to detect a humoral immune response to an infectious agent ofinterest in potentially exposed subjects, to assess vaccinationprotocols or in the development of vaccines. Examples of theseapplications include, but are not limited to the detection of particularstrains of Flaviviruses such as DEN or WNV in human blood, or in insectvectors, or detection of circulating antibodies directed against aparticular strain of DEN prior to commencement of a vaccination program.With the appropriate recognition elements, the assay can also be used todetect the presence of non-pathogen 1-TM receptor ligands such asepidermal growth factor (EGF).

Accordingly, the present invention provides for lipoparticles comprisingat least one fusion protein, wherein the fusion protein comprises atleast one binding domain, at least one transmembrane domain, and atleast one reporter domain.

As used herein, the term “binding domain” refers to a domain that ispresent in the fusion protein that is capable of binding to a ligand.The ligand can be a peptide, protein, fragment of a protein, a nucleicacid molecule (e.g. RNA or DNA), small molecule, hormone, antigen,pathogen (e.g. virus or bacteria), and the like. In some embodiments,the binding domain is an antibody binding domain. An antibody bindingdomain can come from any protein including, but not limited to ProteinA, Protein G, Protein M, or Protein L. In some embodiments, the bindingdomain comprises a ligand-binding portion of a cellular membraneprotein. As used herein, the term “antibody binding domain” refers to adomain that can bind an antibody.

In some embodiments, the fusion protein comprises a transmembrane domainthat spans the lipid bilayer once, twice, three times, four times, fivetimes, six times, or seven times. In some embodiments, the transmembranedomain comprises a membrane anchor. In some embodiments, the membraneanchor comprises about 3, about 4, about 5, about 6, about 7, about 8,about 9, or about 10 amino acid residues. In some embodiments, themembrane anchor comprises at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, or at least 10 amino acidresidues. In some embodiments, the membrane anchor comprises a lipid ora lipid modification. In some embodiments, the membrane anchor comprisesamino acid residues and a lipid or lipid modification. By “lipidmodification” it is meant to refer to a protein that is modified by alipid that allows the protein to be anchored to the membrane. In someembodiments, the lipid modification comprises a covalent attachment of alipid to the protein. In some embodiments, the lipid modification isnon-covalent (e.g. ionic bond, hydrogen bond, and the like). In someembodiments, the membrane anchor is glycosylphosphatidylinositol (GPI).

As used herein, the “reporter domain” refers to a domain in the fusionprotein that can be used to generate a signal that can indicate bindingto the binding domain of the fusion protein. The reporter domain canhave any activity that can indicate binding. In some embodiments, theactivity of the reporter domain comprises enzymatic activity orfluorescent activity. In some embodiments the reporter domain generatesa signal that can be used in a calorimetric, bioluminescent, orchemiluminescent assay. In some embodiments, the reporter domaincomprises a fluorescent protein. In some embodiments, the fluorescentprotein is CFP, YFP, GFP, dsRED, or BFP. In some embodiments, thereporter domain comprises secreted alkaline phosphatase activity,luciferase, chloramphenicol acetyltransferase, β-glucuronidase orβ-galactosidase activity. In some embodiments, the reporter domain ismonomeric.

In some embodiments, the reporter domain is inactive unless it is incontact with a complementary inactive form. Non-limiting examples ofsuch domains include the kinase domains of the EGF receptor (EGFR), theplatelet-derived growth factor receptor (PDGFR), fibroblast growthfactor receptors (FGFRs), the erythropoietin (EPO) receptor, and thegrowth hormone receptor. These kinase domains are inactive until theyare phosphorylated during homodimerization, which is facilitated by thebinding of ligand to the extracellular domain of the receptor.

In some embodiments, a lipoparticle comprises a first fusion protein anda second fusion protein, wherein each fusion protein comprises at leastone binding domain, at least one transmembrane domain, and at least onereporter domain. In some embodiments, the first and second fusionproteins comprise two different reporter domains. In some embodiments,the first and second fusion proteins comprise the same or differentbinding domains, transmembrane domains, reporter domains, andcombinations thereof.

In some embodiments, the reporter domains are capable of FRET or BRET.Examples of domains that are capable of FRET or BRET include, but arenot limited to, CFP, YFP, and the like. Accordingly, in someembodiments, the first reporter domain comprises CFP and the secondreporter domain comprises YFP or vice versa.

In some embodiments, the first and second fusion proteins comprise anantibody binding domain and comprise one or more antibody-likemolecules. As used herein the term “antibody-like molecule” refers to amolecule that is an antibody or a fragment of an antibody. In someembodiments, an antibody-like molecule comprises a monoclonal antibody,a polyclonal antibody, an affinity-purified polyclonal antibody, a Fabfragment derived from a monoclonal antibody, an immunoglobulin-fusionprotein, a single chain Fv, an Fc-fusion protein, or combinationsthereof.

In some embodiments, a fusion protein comprises two differentantibody-like molecules that each recognize different epitopes on thesame protein. In some embodiments, a fusion protein comprises twodifferent antibody-like molecules that each recognize different epitopeson different proteins.

In some embodiments, a lipoparticle comprises a first fusion proteincomprising an antibody binding domain and a second fusion proteincomprising a ligand-binding portion of a cellular membrane protein.

In some embodiments, the binding domain of a first and/or second fusionprotein recognizes different epitopes on the same protein or recognizesdifferent epitopes on different proteins.

The present invention also provides for sensors comprising alipoparticle comprising at least one fusion protein, wherein the sensoris capable of detecting the signaling activity of a cellular membraneprotein. In some embodiments, the signal is detectable in the absence ofa living cell.

“Living cell” refers to any cell that is capable of cell division orcontains a nucleus. A “living cell” also refers to a cell that hasactive metabolic machinery (e.g. mitochondria). In some embodiments, thecellular membrane protein is a seven transmembrane protein or a singletransmembrane domain. In some embodiments, the cellular membrane proteinis a GPCR.

The lipoparticle comprising the fusion proteins can be used to detectthe presence of an antigen. Accordingly, the present invention providesmethods of detecting the presence of an antigen comprising contactingthe antigen with at least one lipoparticle and detecting the signal fromsaid lipoparticle. In some embodiments, the lipoparticle comprises atleast one fusion protein comprising at least one binding domain, atleast one transmembrane domain, and at least one reporter domain.Lipoparticles can be constructed with binding domains that are specificfor different antigens which would allow the lipoparticles to be used todifferentiate between antigens. Lipoparticles can also be constructedthat contain one or more fusion proteins, wherein each fusion proteincomprises a different binding domain with a different reporter domain.For example, a lipoparticle can comprise a first and second fusionprotein, wherein the first fusion protein has a binding domain for anepitope of Dengue Virus (DEN) E protein linked to a reporter domaincomprising CFP and the second fusion protein comprises a binding domainto a different epitope of DEN E protein linked to a reporter domaincomprising YFP. Therefore, when an antigen is contacted with alipoparticle comprising the first and second fusion proteins, thedetection of CFP and YFP FRET activity will indicate the presence orabsence of DEN. In some embodiments, the antigen is contacted with agroup of lipoparticles, wherein the group of lipoparticles comprisesdifferent fusion proteins comprising different binding domains linked todifferent reporter domains. A “group of lipoparticles” comprises morethan one lipoparticle. In some embodiments, a “group of lipoparticles”comprises at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least twenty, at least thirty, or at least fifty lipoparticlescomprising different fusion proteins. In some embodiments, a “group oflipoparticles” comprises two, three, four, five, six, seven, eight,nine, ten, twenty, thirty, or fifty lipoparticles comprising differentfusion proteins. In some embodiments, the lipoparticles are an array oflipoparticles.

As used herein the term “binding domain linked to a reporter domain”refers to an association between the binding domain and the reporterdomain in the fusion protein. This linkage can occur directly by fusingthe binding domain to the reporter domain or can be done indirectlythrough an intervening sequence such as, for example, a transmembranedomain. A “different binding domain linked to a different reporterdomain” refers to binding domains that bind to either different antigensor different epitopes and that each binding domain is linked to adifferent reporter domain. In some embodiments, as a non-limitingexample, if there is a first and a second fusion protein, wherein thefirst has a binding domain specific for HIV and the second has a bindingdomain for WNV, the first fusion protein will have a reporter domainthat is different from the second fusion protein so that the activitiescan be differentiated and thus, the antigens can also be differentiatedfrom one another based on the activity of the reporter domain.

As used herein, “antigen” refers to a composition comprising at leastone pathogen protein, at least one whole-pathogen organism, at least onesecreted cellular peptide, at least one immobilized protein, at leastone tissue section, at least one cell, at least one ligand, at least onesmall molecule, at least one hormone, or at least one antibody. In someembodiments, that pathogen protein is a pathogenic protein or peptidefrom an agent selected from: picomavirus family, calcivirus family,togavirus family, flariviridue family, hepatitis C virus, coronavirusfamily, rhabdovirus family, filoviridue family, paramyxovirus family,orthomyxovirus family, bungvirus family, arenavirus family, reovirusfamily, retrovirus family, papovavirus family adenovirus family,parovirus family, herpesvirus family, poxvirus family, hepadnavirusfamily, hepatitis delta virus, pathogenic gram positistive cocci,pathogenic gram-negative cocci, pathogenic enteric gram-negativebacilli, pathogenic anaerobic bacteria rickettsia, mycoplasms,Chlamydia, pathogenic protozoans, and pathogenic helminthes. In someembodiments, the antigen is a pathogenic protein or peptide from anagent causing a disease selected from HIV-1 infection, HIV-2 infection,SIV infection, Hepatitis C, Hepatitis B, HPV infection, HSV infection,HTLV infection, HCMV infection, WNV infection, Dengue Virus infection,spirochete B. bugdorferi, rabies, gastroenteritis, foot and mouth,dengue, yellow fever, encephalitis, common cold, bronchitis,encephalymylitis, haemorrhagic fever, mumps, new Castle disease, caninedistemper, Rift Valley fever, Nairobi sheep disease, Lassa Fever,Colorado Tick fever, Lebombo, equine encephalosis, blue tongue,smallpox, anthrax, and cowpox. In some embodiments, the antigen is acomposition that comprises more than one antigen, a group of antigens,or an array of antigens. In some embodiments, the composition comprisesone, two, three, four, five, six, seven, eight, nine, ten antigens. Insome embodiments, the composition comprises at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten different antigens.

In some embodiments, the presence of an antigen is detected byfluorescence, resonance energy transfer, luminescence, or total internalreflectance fluorescence. Detection can be done with any deviceincluding, but not limited to a microfluidic device, a flow cell, aLab-on-a-Chip, a microplate with wells, a 96-well plate, a 384-wellplate, a 1536-well plate, a glass slide, a plastic slide, an opticalfiber, a prism, a flow cytometer, a microscope, a fluorometer, aspectrometer, or a CCD camera.

In some embodiments, the method detects an infectious agent, areceptor's ligands, or a pathogen-specific antibody. The methods canalso be used for pathogen screening, disease diagnosis, drug screening,epidemiological surveys, or food contamination assessment.

In some embodiments, the method further comprises contacting at leastone lipoparticle with a control antigen and generating a signal inresponse to the control antigen relative to another antigen that isunknown or needs to be characterized. If the signal generated ordetected are the same, this would indicate that the unknown antigen isthe same as the control. If the signal generated or detected by thecontrol and unknown antigen are different, this would indicate that thetwo antigens are not the same. Therefore, this method can be used toidentify or confirm an antigen as being similar or different to acontrol (e.g. known) antigen. This can be use, for example, to rule outpathogenic agents as the cause of a disease, disorder, epidemic, orcontamination. This can also be used in conjunction with an array orgroup of control antigens so that more than one antigen or pathogen canbe ruled out or in at a time, which would be important in saving time toidentify the cause of a disease, disorder, epidemic, or contamination.

In some embodiments, the present invention provides devices comprisingat least one lipoparticle that can be used to identify the presence oridentity of an antigen. In some embodiments, the device comprises afluorometer, which can be, for example, a portable fluorometer weighingunder 50 pounds, under 20 pounds, under 5 pounds, or under 2 pounds.

The present invention also provides other uses of lipoparticles.Lipoparticles can be used for the development of nanometer scalebiological probes, known as LipoProbes. In some embodiments, LipoProbesuse lipoparticle structure as a vehicle for the assembly of a targetingand/or reporter combination. In some embodiments, a LipoProbe comprisesa lipoparticle, which comprises surface targeting molecules (e.g.membrane-bound antibodies or antibody-fragments), and/or reportingmolecules (e.g. fluorescent proteins fused to the Gag protein). In someembodiments, the lipoparticle enables the assembly of multiple targetingand reporting systems individually or in combination. These can becoupled to the lipoparticle surface, incorporated within the lipid orprotein components of the membrane, linked to structural proteins suchas Gag, and encapsulated in soluble form within the lipoparticlemembrane-bound cavity. In addition, lipoparticles can incorporatemolecular constructs to facilitate LipoProbe handling, and to modifytargeting, sensing and reporting functions (such as channels for ionsensitivity or biotin tags for manipulation), as well as effectormolecules capable of perturbing micro-environmental systems (e.g.enzymes). LipoProbes are suitable for a vast range of applications,including monitoring protein-protein interactions such as ligandbinding, structural mapping, cell signaling, detection ofmicro-environmental conditions such as pH or ion concentration, andtracking lipid interactions such as during viral-host fusion. LipoProbescan exploit a variety of detection techniques, including innatefluorescence and luminescence, enzyme-mediated detection, FRET,polarization, and TIRF, and can be monitored using a diverse array ofdetection modalities including microscopy, micro-plate, cuvettes, andflow cytometry. They are suitable for use with a range of target formats(e.g. in vitro, ex vivo, or in vivo). LipoProbes have utility in avariety of health and biological science fields, including lead compoundidentification for drug discovery, pharmaceutical development,diagnostics and preventive medicine, cell biology, virology andproteomics. In addition, LipoProbes can provide valuable adjunctfunctions in research and development programs, including proteinproduction and purification (e.g. for structural studies), isolation ofligands and drugs, monoclonal antibody production, hybridoma screening,and epitope mapping.

The flexibility of LipoProbes lies in the variety of methods by whichlipoparticles can be modified to include active molecules. Lipoparticlescan be endowed with a variety of interactive targeting, reporting, andeffector functions by modifying the structure and composition ofmembrane lipids, membrane proteins, core proteins, and by altering thebiochemical characteristics of the lipoparticle interior.

Lipoparticle Modifications: Creating LipoProbes

Modification of the Lipoparticle Surface

Lipoparticles can be modified by coupling them to molecules or complexstructures via reactive groups exposed on the lipoparticle surface.Lipoparticle membranes are derived from cells, and so incorporateproteins and carbohydrates that either naturally, or can be chemicallymodified to, possess reactive molecular groups, such as amines, carboxylgroups, carbonyls, and sulfhydryl groups. These groups can be recognizedand acted upon directly by binding proteins. For example, lectins, suchas concavalin A (ConA), wheat germ agglutinin (WGA), and legume lectins,bind oligosaccharide groups (e.g. galactose, acetyl-D-glucosaminyl,acetylgalactosaminyl, mannopyranosyl, and galactopyranosyl residues)commonly contained in glycoproteins, proteoglycans, and glycolipids.These lectins can, in turn, also be modified, such as biotinylated WGAlectin. Cholera toxin subunits (A and B) bind galactosyl moieties. Lipidmoieties, such as fatty acids may also be directly targeted by bindingproteins, such as I-FABP, a rat intestinal fatty-acid binding protein.Reactive groups may also be chemically modified by cross-linking agents,such as amine-reactive imidoesters and N-hydroxysuccinimide(NHS)-esters, sulfhydryl-reactive maleimides, pyridyl disulfides andhaloacetyls, carbonyl-reactive hydrazides, or carboxyl-reactivecarbodiimides. Such binding proteins and cross-linking agents arecommonly conjugated to functional molecular species. These methods canbe used to link functional molecules, such as fluorescent ornon-fluorescent reporters, enzymes, binding and targeting proteins (e.g.antibodies), and complex structures such as beads or other solidsubstrates, magnetic particles, gold particles and radioactivesubstances to lipoparticle surfaces. These modifications can conferunique interactive properties on lipoparticles that enable localizationto targets or substrates, visual detection (e.g. fluorescence), anddetection by MRI, electron microscopy, radiography, or PET.

The lipoparticle can be modified to accommodate any of theseinteractions, such as its use as a probe on cells. Modifications caninclude treatment with a protease such as proteinase K or trypsin, aglycosidase such as EndoF or EndoH, or mixing with a blocking reagentsuch as BSA, serum, PEI, Pluronics, an RGD-containing peptide, orpolylysine. In some embodiments, the lipoparticle comprises a membraneprotein resistant to such treatments, for example a ZZ-TM fusion proteinengineered to lack any trypsin cleavage sites.

In some embodiments, the virus, virus-like particle, or lipoparticlecomprises at least one of a radioactive molecule, a magnetic substance,a paramagnetic substance, a biotinylated molecule, an avidin-likemolecule, gold, or combinations thereof and optionally a fluorophore.

Modification of the Lipids within the Lipoparticle Membrane

The lipid composition of cell membranes is an important determinant ofsuch properties as fluidity, permeability, and electrical potential.Alterations in the nature and quantity of membrane lipids can influencethe formation of specialized micro-structures (e.g. microvilli) andmembrane protein functions. The lipid composition of lipoparticles canbe influenced by depletion of specific lipid constituents (such ascholesterol or sphingolipids), by addition of lipid moieties (e.g.insertion of amphiphilic molecules such as phospholipids and otherglycerol-derived components), and by modification of the chemical andstructural nature of naturally incorporated lipids (e.g. modification ofthe saturation state of acyl chains, stabilization using lipid fixativessuch as osmium tetroxide). Modification of membrane lipids present inthe lipoparticle structure can be achieved during lipoparticleproduction by inducing metabolic alterations in producer cells, orpost-production by chemical treatment of lipoparticles. Lipolyticenzymes (e.g. phospho- or galactolipases, acyl hydrolases), milddetergents (e.g. polyethylene glycol, and lubrols such as Lubrol W), andlipid-complex forming agents (e.g. cyclodextrins) can be used toselectively or non-specifically deplete lipids from lipoparticlemembranes. The specific lipid composition of lipoparticle membranes canbe altered by liposome-mediated lipid enrichment, or by treatment withfatty acids and fatty-acid analogs (e.g. pyrenes, undecanoic acid,parinaric acid) or phospholipids (e.g. phosphoinositides,phsophocholines, phosphoethanolamines). A variety of other lipid analogsand lipophilic molecules, such as sphingolipids, steroids,triacylglycerols, octadecyl rhodamine, lipophilic fluoresceins,coumarins, dialkylcarbocyanine probes, and dialkylaminostyryl probes,could similarly be incorporated directly into the lipid bilayer of thelipoparticle membrane. These lipid molecules may possess chemicalmodifications of acyl chains or other molecular groups, or may beconjugated to molecular groups or complex structures, conferring thecapacity for target interaction (e.g. biotin, avidin, antibodies,enzymes), reporter functions (e.g. fluorescent and/or luminescent dyessuch as Nile Red, Rhodamine 421, di-4-ANEPPS, Oxonol VI, DiSC3(5), diI,di-BAC4), other effector or detector functions (e.g. leukotrienes,prostaglandins, eicosanoids, thromboxanes), or for altered lipoparticlelongevity or tissue processing (e.g. PEG).

Additional changes in the lipid composition of the lipoparticle can alsobe introduced. For example, purified lipids (e.g. PE) can be added topurified lipoparticles and allowed to partition into the lipoparticle.Similarly, cholesterol can be depleted from the lipoparticle by usingMDBC, which soaks up cholesterol from cell membranes. In addition,lipids can be cross-linked to toughen the lipid bilayer of thelipoparticle.

Modification of the Proteins within the Lipoparticle Membrane

Lipoparticles can incorporate a variety of membrane proteins, includingreceptors, ion channels, and transporters, in their native,functionally-active forms. However, it is also possible to producelipoparticles containing structurally-modified versions of membraneproteins. Plasmids containing sequences for two or more proteins can beused to generate lipoparticles incorporating membrane proteins fused toa variety of other functional proteins. These fusion partners can confertarget interaction properties (e.g. enzymes, antibody-binding proteins),reporter properties (e.g. fluorescent proteins such as GFP, YFP, CFP anddsRed; enzymes such as luciferase, alkaline phosphatase, horseradishperoxidase, beta-galactosidase, and other oxidases, kinases andproteases). Membrane proteins can also be modified post-lipoparticleproduction. Fixation of complex biological structures, such as cells andtissues, with aldehyde solutions such as formaldehyde, paraformaldehyde,and glutaraldehyde results in the formation of methylene bridges betweenprotein nitrogen atoms. The cross-linking preserves protein structuralintegrity, and forms an insoluble matrix that traps carbohydrates andlipids without altering their chemical compositions. Fixation oflipoparticle protein constituents in this way can alter their longevityand behavior in a number of applications such as immunoprobing.

Modification of the Lipoparticle Core Proteins

Normal retroviruses express a core polyprotein consisting of a mainstructural component, Gag, fused to enzymatic proteins, Pol. However,Gag is the one retroviral protein necessary, and sufficient, forlipoparticle production. The Pol polyprotein sequence can be substitutedwith a variety of alternative genes, including those for fluorescentreporter proteins such as GFP, YFP, BFP, CFP, DsRED, AsRED, AmCyan,HcRed, ZsGreen, ZsYellow or variants thereof (Bennett, et al. (1991), JVirol, 65:272-80, McDonald, et al. (2002), J. Cell Biol., 159: McDonald,et al. (2003), Science, 300:1295-7, Weldon, et al. (1990), J Virol,64:4169-79), and non-fluorescent proteins (e.g. esterases, proteases,kinases, alkaline phosphatase, peroxidase, beta-lactamase, luciferase).Approximately 1,000-2,000 Gag proteins form the structural core of eachlipoparticle (Knipe, et al. (2001)), so proteins fused to Gag are highlyrepresented. They can confer reporter functions (e.g. fluorescentproteins), protein manipulation functions (e.g. HA epitope tags), orother modifying functions (e.g. enzymes) to lipoparticles. Additionally,Gag can act as a protein production scaffold for the large-scaleproduction and purification of partner proteins for structural studies.

The Gag fusion protein may also comprise a fusion between Gag and thebinding portions of an antibody, for example a single-chain region Fv.The specificity of the antibody can be used to link the Gag fusionprotein with other proteins during lipoparticle production, which can beused in the purification of the other protein.

In some embodiments, more than one enzyme is incorporated into alipoparticle simultaneously. In some embodiments, the more than oneenzymes could act synergistically on a substrate that requires both oftheir activities. Thus, lipoparticles can act as a reaction center,providing all enzymes necessary for a certain reaction. If provided witha substrate and/or cofactors, entire enzymatic activities can berecapitulated.

Modification of the Biochemical Characteristics of the LipoparticleInterior

Although the biochemical features of retroviruses are poorly understood,the characteristics of their membrane-limited space bear someresemblance to that of the host-cells from which they were derived. Itis possible to alter these features in lipoparticles in order to conferspecific sets of functional properties. The enrichment or depletion ofspecific ions (e.g. Ca⁺⁺, K⁺, Na⁺, Mg⁺⁺, and the like), and theinclusion of small molecules (e.g. nucleic acids such as ATP and GTP andtheir analogs), of proteins (e.g. enzymes, nucleic acid bindingproteins), of dyes and reporters (e.g. water soluble fluorescentmolecules), or of complex structures (e.g. quantum dots) within thelipoparticle structure are possible. The lipoparticle membrane, althoughgenerally considered impermeable, will allow the equilibration of smallspecies such as ions or small nucleic acid binding molecules (e.g.YOYO-1) across it over time, allowing adjustment of lipoparticle ionconcentration by treatment in appropriate buffers. Some water-solublemolecules can be allowed to cross lipid membranes by acetoxymethylesterification (e.g. AM-PBFI, calcein-AM, Fura-2-AM, SNARF-1-AM,AM-SBFI, DAF-FM), after which they are trapped within themembrane-delineated space by enzymatic removal of ester groups. Largermolecules and structures that cannot cross lipid membranes, and are notamenable to chemical modification such as esterification (e.g. quantumdots, Phen-Green, lucigenin, OPA, radioactive particles, paramagneticbeads, Raman probes (DSBB), gold particles) can be loaded intolipoparticles using temporary poration or permeabilization of thelipoparticle structure. Temporary poration or permeabilization can beachieved by such treatments as electroporation, exposure to chemicals orproteins (e.g. streptolysin-O, aerolysin, maltoporin, P2X7, melittin),mechanical stress (e.g. sonication, vortex mixing), and the like.Permanent poration of lipoparticle membranes could also be performed inorder to allow interaction of trapped molecules (such as reporters) withsmall molecules in the microenvironment. Any other method of temporaryporation or permeabilization can also be used.

Caged Reporters

Caged molecules are not active until stimulated in a defined manner,(e.g. by exposure to UV light). Such molecules can be loaded intolipoparticles to confer specific activation characteristics to thelipoparticle probe. Caged molecules include caged versions ofnucleotides and phosphatases (e.g. ATP, ADP, cAMP, cGMP, GTP,GTP-gammaS, GDP, IP3, H+(pH), PO4, ADP-ribose), ion scavengers (e.g.EGTA, EDTA, Diazo-2, thapsigargin, terbium), neurotransmitters (e.g.carbachol, GABA, NMDA, L-glutamic acid), fluorescein, fluorescent IP3,nucleotides (e.g. GTP-gammaS), amino acids, L-glutamic acid, carbachol,gamma-aminobutyric acid, NMDA, O-GABA, and the like.

Lipophilic Fluorophores

Fluorophores can also be incorporated into lipoparticles usingfluorescent dyes. In some embodiments the fluorescent dye is ahydrophobic dye. A hydrophobic fluorescent dye is a dye that fluorescesmore strongly in a non-aqueous environment. In some embodiments, thehydrophobic fluorescent dye does not appreciably fluoresce in an aqueousenvironment. In some embodiments, the hydrophobic fluorescent dye doesnot fluoresce in an aqueous environment. As used herein, the term “doesnot appreciably fluoresce in an aqueous environment” refers to thefluorescent property of a compound (e.g. dye). In some embodiments thefluorescence of a compound that does not appreciably fluoresce in anaqueous environment is about 10% less, about 20% less, about 30% less,about 40% less, about 50% less, about 60% less, about 70% less, about80% less, about 90% less, about 91% less, about 92% less, about 93%less, about 94% less, about 95% less, about 96% less, about 97% less,about 98% less, about 99% less, or 100% less in an aqueous environmentthan it fluoresces in a non-aqueous environment. Examples of non-aqueousenvironments include, but are not limited, a lipid bilayer, cellmembrane, and the like. Fluorescent dyes can be incorporated intoparticles as described in U.S. Application Ser. No. 60/498,755. In someembodiments, dyes that fluoresce strongly in both aqueous and lipidenvironments, the unincorporated dye is separated from the lipoparticlesprior to visualization, for example, by a gel filtration spin column.

Examples of dyes that bind and/or label lipids include, but are notlimited to, Amphiphilic dyes; DiA; 4-Di-10-ASP; FASTDiA; FM 1-43; FM4-64; FM 5-95; NBD; TMA-DPH; TMAP-DPH; ANS; MBDS; BADS;4-amino-4′-benzamidostilbene-2,2′-disulfonic acid, disodium salt (MBDS);1-anilinonaphthalene-8-sulfonic acid (1,8-ANS);2-anilinonaphthalene-6-sulfonic acid (2,6-ANS); bis-ANS(4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt),(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene(BODIPY® 665/676); 1,10-bis-(1-pyrene)decane;1,3-bis-(1-pyrenyl)propane; Dapoxyl® sulfonic acid sodium salt;4-(dicyanovinyl)julolidine (DCVJ);6,8-difluoro-4-heptadecyl-7-hydroxycoumarin (C17DiFU);4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene(BODIPY® 493/503);4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY®505/515) 4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole (NBDdihexadecylamine);4-(N,N-dimethyl-N-tetradecylammonium)methyl-(7-hydroxycoumarin) chloride(U-6); 1,6-diphenyl-1,3,5-hexatriene (DPH);5-dodecanoylaminofluorescein; 6-dodecanoyl-2-dimethylaminonaphthalene(laurdan); fluorescein octadecyl ester; 4-heptadecyl-7-hydroxycoumarin;5-hexadecanoylaminofluorescein;6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalenechloride (patman); Nile red; Di-4-ANEPPS; Di-8-ANEPPS;5-octadecanoylaminofluorescein;N-octadecyl-N′-(5-(fluoresceinyl))thiourea (F18); octadecyl rhodamine Bchloride (R18); 3-(4-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid(DPH propionic acid);N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammoniump-toluenesulfonate (TMAP-DPH); N-phenyl-1-naphthylamine;6-propionyl-2-dimethylaminonaphthalene (prodan); 1-pyrenebutanol;1-pyrenenonanol; 1-pyrenesulfonic acid, sodium salt;2-(p-toluidinyl)naphthalene-6-sulfonic acid, sodium salt (2,6-TNS);1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatrienep-toluenesulfonate (TMA-DPH); diI; diS; diO; Oxonol VI; JC-1; DiSC3(5);Rhodamine 421; and the like.

LipoProbe Function and Applications

In some embodiments, these probes described herein can be used to mapcell and tissue structures both in vitro and in vivo, as well as todetect single or multiple cell signaling events. Modifying molecules canalso be incorporated into the LipoProbe to alter its targeting,biophysical properties, and sensitivity. Table 10 outlines some of theembodiments of the LipoProbe and some of their applications.

TABLE 10 Examples of potential LipoProbe components and theirapplications. Applications Proteins ZZ-TM This transmembrane anchoredprotein can bind the Fc portion of user-specified antibodies, enablingtargeted localization of labeled LipoProbes. CXCR4, CD4, DC- Thesemembrane proteins bind cognate ligands, such as the chemokine SDF-1SIGN, Fas, TNFR (CXCR4), Fas Ligand, TNF, and HIV Envelope (CD4, CXCR4,DC-SIGN), enabling interactions among membrane proteins in their native,lipid-anchored form to be detected. Extracellular proteins such asintegrins, TNF receptor family members, and immunoadhesins, are ofspecial interest because they involve interactions between membraneproteins on opposing lipid surfaces. Protein Kinase C (PKC), Bothsoluble (PKC) and membrane-bound (BACE) enzymes can be incorporatedbeta-site APP-cleaving into the LipoProbe and used as effectors on theirnatural targets (RXXS/T enzyme (BACE) phosphorylation motifs andamyloid-beta precursor protein (APP), respectively). Reporters BFP, CFP,GFP, YFP, Fluorescent proteins can be used as simple fluorescent tags,to detect specific dsRed conditions (e.g. pH-sensitive GFP), or fordetecting binding interactions (e.g. FRET). di-4-ANEPPS, di- Lipophilicmolecules readily incorporate into the lipid membrane of LipoProbes.BAC4, Oxonol VI Many respond to environmental conditions (pH, membranepotential) during phagocytosis or in cellular structures (e.g.synapses). Fura-2, Indo-1, quantum Hundreds of soluble reporters (somein AM-ester form) can be incorporated that are dots responsive tochanges in ion concentration or that exhibit incomparable fluorescenceand longevity, creating probes of Ca++ and methods of tracking thedestinations of infectious viruses (quantum dots). ³H, ³⁵S, paramagneticRadioactive tracers and substances that can change electromagneticproperties can beads, gold nanospheres be incorporated into LipoProbesfor diagnostic imaging techniques such as MRI and PET ModificationsTRPV1, TRAP6, CFTR, The incorporation of ion channels that open inresponse to specific stimuli (e.g. Shaker-K Ca⁺⁺-channel TRPV1 opens inresponse to heat) and gate select ions allows the LipoProbe to act as asensor of these stimuli and to link incorporated reporters to the ionchanges (e.g. Ca⁺⁺ responsive Fura-2). Biotin-PE, PEG-PE Partitioning offunctionalized lipids into the LipoProbe can allow targeting (biotin) orenhanced longevity (PEG)

Numerous alternative reporter proteins and dyes can be incorporated intoLipoProbes using the methods tested here, including, but not limited to:Visual reporters (e.g. beta-lactamase, beta-galactosidase, HRP, alkalinephosphatase, luciferase); Catalytic enzymes (e.g. kinases, phosphatases,proteases, oxidases); Fluorescent proteins e.g. (GFP, BFP, CFP, YFP,dsRED, numerous variants); Lipophilic dyes (e.g. Nile Red, Rhodamine421, Oxonol VI, DiSC3(5), diI); Water-soluble probes (e.g. Phen-Green(heavy metals), lucigenin (Cl⁻), OPA (cyanide)); AM-ester conjugates(SNARF-1 (pH), Fura-2 (Ca⁺⁺), SBFI (Na⁺), DAF-FM (NO)); Non-visualreporters (e.g. radioactivity, paramagnetic beads, Raman probes (DSNB),gold).

Many of these alternative reporters can improve LipoProbe sensitivity(e.g. luminescent proteins, FRET pairs, Raman assays), extend LipoProbeapplications (PET, MRI), and/or improve the functional capabilities ofLipoProbes (e.g. by sensing environmental conditions such as membranepotential, pH, and heavy metals). Because many second-generationdetection techniques rely on the simultaneous activity of multiplereporters, LipoProbes allow incorporation of reporters by multipleindependent methods, any of which can be combined.

In some embodiments, the lipoparticle combines individual reporters intoan integrated detection system. The reporters are not only in closeproximity within the lipoparticle, but can be linked so that theiractivation must occur sequentially. Reporters that can be incorporatedinto the lipoparticle include small molecules, proteins, ion channels,and quantum dots. Unlike liposomes reconstituted withdetergent-solubilized ion channels, lipoparticles can be used toincorporate a wide variety of ion channels without the requirement fordetergent solubilization. In addition, soluble protein reporters areincorporated as structural elements of the lipoparticle, ensuring highcopy number and ease of production.

The reporters within a lipoparticle can be chosen to respond to the sameor different signals. In some embodiments, the lipoparticles responds toabout 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,or about 10 different signals. In some embodiments, the lipoparticleresponds to least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 10 differentsignals. In some embodiments, the lipoparticles respond to only 1signal. In some embodiments, the lipoparticles responds to about 1-100,about 1-50, about 1-40, about 1-30, about 1-20, about 1-10, about 1-5,about 2-10, about 2-5 signals. Thus, the lipoparticle Sensor can havethe capability to monitor multiple points along a signal transductionpathway. When a lipoparticle detects the same stimulus, multiplereporters can provide increased sensitivity of detection over a greaterrange and increased detection specificity. When they detect differentstimuli, multiple reporters can provide the ability to monitor multipleevents within a signal transduction pathway. The reporters can also bedesigned to simultaneously detect different events from multiplesignaling pathways, allowing complex cellular responses to be monitored.The use of a combinatorial detection system is especially important incells where low signal-to-noise ratios and interference by backgroundcellular pathways are a significant concern in the detection of realsignals. The lipoparticle makes this sensing system possible byconfining sensors to a nanometer-sized probe, which, by modifying thelipoparticle with specific external antibodies or proteins, can betargeted to defined subcellular locations.

Selective modifications can be introduced to the lipoparticle to enableit to be used in more harsh environments such as in cells, in animals,and with detergents. Modifications include PEGylation, polymerization oflipids, fixation of lipids, fixation of proteins, enzymatic digestion ofexterior proteins, enzymatic removal of exterior carbohydrates, andpre-incubation with molecules that block non-specific binding.

Because the lipoparticle is based on a virus structure, the LipoProbealso has direct application to the study of viruses. The ability toincorporate more robust probes into viruses (e.g. quantum dots) andprobes with more meaningful signals (e.g. pH- or membranepotential-sensitive dyes) could allow a number of new insights intoviral infection processes.

The lipoparticle Sensor is a sensing system, combining existingreporters within a nanometer-scale vehicle with unique sensingproperties. In some embodiments, cells microinjected with thelipoparticle Sensor can be monitored for both fluorescence andluminescence in real-time using microscopic imaging. Cells can bemonitored while stimulating with 1) heat (heating to 43° C. activatesTRPV1) and 2) the GPCR agonist TRAP-6 (initiates Ca⁺⁺ flux within thecell by stimulating the thrombin receptor PAR-1). When the cells arestimulated with heat, the Ca⁺⁺-ion channel on the lipoparticle Sensor isactivated and allows the passage of Ca⁺⁺-ions into the lipoparticle. Insome embodiments, the ion channel is activated and Ca⁺⁺ flows into thelipoparticle and activates the Ca⁺⁺-ion reporters, which can be detectedby changes in fluorescence and luminescence. A simultaneous change inboth reporters permits a high degree of confidence in the signal.

Target Interaction and LipoProbe Localization

Among the lipoparticle modifications that confer utility as probes arethose that enable interaction with specified targets, for targetrecognition and detection, and/or for directing LipoProbes to specificlocations. Target interactive-components can include molecules andcomplex structures (e.g. biotin, avidin, streptavidin, WGA beads,enzymes) linked covalently or otherwise to the lipoparticle surface, orconjugated to modified lipid molecules within the lipoparticle membrane.Functional membrane-associated proteins, either native, or modified,such as receptors (e.g. CXCR4, CCR5), antibody binding proteins (ProA,ProL, ProG), enzymes (e.g. protein kinase C, beta-site APP-cleavingenzyme), and ion channels (e.g. TRPV1, CFTR, Shaker) can be incorporateddirectly into the lipoparticle membrane. Active proteins (e.g.carboxylases) can be fused to membrane proteins or to viral structuralproteins, while organic chemicals (e.g. ion chelators, active smallmolecules such as nucleic acid binding compounds, pH-sensitivemolecules, oxygen-reactive species) can be incorporated within thelipoparticle interior. Target-interacting components can enableLipoProbes to interact with receptor ligands, with antibodies, withspecific antigenic epitopes, with specific lipids or carbohydrates, withions (e.g. H⁺, K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺), with reactive molecules in solution(e.g. reactive oxygen species), or with immobilizing substrates (e.g.coated or surface-reactive beads or solid substrates).

Methods for Visualizing and Detecting Lipoprobes and their Interactions

Lipoparticles can be modified to enable them to be visualized ordetected by emission of fluorescence and luminescence, visible light,radioactivity, or other electromagnetic emissions. These reporters canbe linked to the lipoparticle surface or incorporated into the lipidmembrane (e.g. fluorescent beads, fluorescent lipid molecules,lipid-soluble dyes), fused to or otherwise attached to integral membraneproteins (e.g. CXCR4-GFP), fused or otherwise attached to structuralproteins (e.g. Gag/luciferase, Gag/GFP), or loaded in soluble form intothe lipoparticle interior (e.g. AM-ester dyes, nucleic acid bindingmolecules such as SYBR green, DAPI, and YOYO-1). Such reporters can beconstitutively active (e.g. GFP, fluorescein, ALEXA), can require someinteraction with targets or co-factors for detection (e.g. luciferase,FRET pairs, polarization reporters), or can require some user-definedintervention (e.g. exposure to ultraviolet light) to enable reportingactivity. These so-called ‘caged’ reporters include caged versions ofnucleotides and phosphatases (ATP, ADP, cAMP, cGMP, GTP, GTP-gammaS,GDP, IP₃, H⁺ (pH), PO4, ADP-ribose), ion scavengers (EGTA, EDTA,Diazo-2, thapsigargin, terbium), neurotransmitters (carbachol, GABA,NMDA, L-glutamic acid), fluorescein, fluorescent IP3, nucleotides(GTP-gammaS), amino acids, L-glutamic acid, carbachol,gamma-aminobutyric acid, NMDA, O-GABA. LipoProbes incorporatingreporters can be detectable using a variety of detection and assayplatforms including, but not limited to conventional or confocalfluorescent microscopy, conventional fluorometry (microtiter plate orcuvette), fluorescent emission polarity shift detection, flow cytometry,and TIRF.

Application-Driven Modification Combinations

LipoProbes can be used for a variety of applications, defined by thespecific combination of incorporated modifications. The LipoProbe is nota reporter in itself, but rather a sensing system, combining existingtarget interaction and reporter components, along with ancillarymodifications that facilitate, modify, or enhance target interaction andreporting functions.

Binding Assays

Based on the ability to detect particles using fluorophores, the presentinvention provides methods of detecting the binding of compounds toparticles. In some embodiments the methods involve detection of thebinding of compounds to lipoparticles. The compound can be any compoundthat is thought to bind to, or it is being tested to determine if itbinds to, the particles including, but not limited to, ligands,peptides, proteins, antibodies, organic chemical compounds (e.g. smallmolecular weight compounds), or inorganic chemical compounds. In someembodiments, the compound comprises a fluorophore or fluorescent label.

The compounds can be contacted with the particle to determine if thecompound can bind to the particle. To determine if the compound can bindto the lipoparticle one can detect the binding through the fluorescenceof bound compound when the compound comprises a fluorophore orfluorescent label. Detection methods include, but are not limited to,flow cytometry, immunofluorescence, sensors, microscopy, and the like.Sensors and using them with particles is described herein and, forexample, in U.S. Application No. 60/491,633. In some embodiments, thecompound is immobilized on the sensor surface. In some embodiments, theparticle is immobilized on the sensor surface.

The present invention also provides methods of identifying compoundsthat bind to the same site as that of a compound that is known to bindto a particle or to a site that prevents the compound known to bind to aparticle by some other mechanism such as steric hindrance or anallosteric change in the particle. This can be detected using theability to detect particles using the present invention. In someembodiments, the method comprises contacting a first compound with aparticle to which a second compound is already bound to or to which itis known that a second compound can bind to, but has not yet beencontacted with the particle. In some embodiments, the first compoundcomprises a fluorophore. In some embodiments, the second compoundcomprises a fluorophore. In some embodiments, the first and secondcompounds comprise different fluorophores. One can detect if the secondcompound is prevented from binding to the particle by the change influorescence that is observed. If only the second compound comprises afluorophore, then a decrease in fluorescence once the first compound isbrought into contact with the particle would indicate that the firstcompound prevents or inhibits the second compound from binding to theparticle. If only the first compound comprises a fluorophore, then anincrease in fluorescence would indicate that the second compound isinhibited or prevented from binding to the particle by the presence ofthe first particle. In some embodiments, the particle comprises afluorophore and the fluorescence can indicate whether or not a compoundis bound to the particle. In some embodiments, when the second compoundis bound to the particle, the particle fluoresces, whereas when thefirst compound binds to the particle the particle does not fluoresce andvice versa. Therefore, the changes in fluorescence can be used todetermine if the first compound inhibits or prevents the second compoundfrom binding to the particle. The changes in fluorescence, eitherassociated with the particle or the compound, can also be used todetermine if the first compound can bind to the particle. Methods ofmeasuring fluorescence are known to those of ordinary skill in the art.

Detection of Protein-Protein Interactions

In some embodiments, the Lipoprobe can also be used to discover andcharacterize protein-protein interactions, using techniques such as farwestern, flow cytometry, and immunostaining. For example, lipoprobescarrying one membrane protein can be used to test binding to cellsexpressing other membrane proteins. Membrane protein pairs can includeICAM/LFA, Fas-FasLigand, class I/class II/CD4/CD8/T cell receptorcombinations, or Notch and Delta, and the like.

In some embodiments, orphan membrane proteins can be used to search forpairing ligands. A lipoparticle comprising an orphan membrane protein isscreened against a sample comprising molecules that may bind to theorphan membrane protein. In some embodiments, the sample is a library ofligands, tissue homogenate, cells, and the like. A ligand that can bindto the orphan protein can be identified by a change in fluorescence ofthe lipoparticle or detectable change in the lipoparticle that wouldindicate that a molecule has bound to the orphan membrane protein.

Flow Cytometry

Another means of detecting the structural integrity of membrane proteinscontained in lipoparticles is flow cytometry. Because flow cytometrymeasures individual events, flow cytometry can be used to quantify thenumber of lipoparticles in a given sample. If a fluorescent marker tothe membrane protein is used, flow cytometry can be used to quantify thenumber of membrane proteins in a given sample. The lipoparticles to bequantified can be made fluorescent using a Gag-GFP fusion protein, alipid dye, a receptor-GFP fusion protein, a secondary antibody bound tothe lipoparticle, or any other method of staining a lipoparticle. Byusing a control (200 nm latex beads) containing a known number ofantibody binding sites, the number of receptors per lipoparticle canalso be determined.

In some embodiments, the lipoparticles are attached to beads before flowcytometry. The beads may be larger in size (e.g. 10 μm) in order tobetter accommodate a flow cytometer detector. In one embodiment, thebeads are fluorescently labeled. In another example, the lipoparticlesare biotinylated and the beads are coated with streptavidin tofacilitate linkage. In another example, the beads are coated with thelectin Wheat Germ Agglutinin (WGA).

Reactive Oxygen Detection

A wide variety of active substances can be delivered to specificlocations by incorporation into targetable LipoProbes. Reactive oxygenspecies, with short (nano- to microsecond) lifespans or limiteddiffusion capabilities, such as singlet oxygen or hydroxyl radicals, canbe delivered with nanoscale precision. Reactive oxygen and nitric oxidespecies react with a wide range of molecules, including NADH, dopa,ascorbic acid, histamine, tyrosine, tryptophan, cysteine, glutathione,nucleic acids, cholesterol, and unsaturated fatty acids, and can bedifficult to distinguish in whole cells. By compartmentalizing themand/or agents for their detection within LipoProbes, specificity ofdelivery, interaction, and detection can be increased considerably.

Nucleic Acid Detection

The lipoparticle may also be incubated with a nucleic acid probe that iscomplementary to a nucleic acid sequence in the particle. In such case,the probe may be fluorescently or radioactively labeled. Hybridizationof the probe with the lipoparticle and detection by, for example,fluorescent microscopy, would be indicative of the nucleic acid sequenceexisting in the nucleic acid occurring in the particle. Such methods maybe useful for diagnostics. Nucleic acid probes can also be used toidentify a specific type of virus. In some embodiments, a nucleic acidsequence is added to a sample containing a virus that will fluoresce ifthe nucleic acid molecule is able to hybridize to the virus. If asequence that is specific to a virus or family of viruses is used thesemethods can used to identify the presence and/or type of virus or virusfamily in a sample. Accordingly, the present invention can be used fordiagnostics of viruses in a sample. Additional components which interactwith nucleic acids, such as the de-stabilizing enzyme RecBCD, could alsobe incorporated into lipoparticles. Nucleic acid-specific dyes used inconjunction with LipoProbes could have application, among other things,in diagnostics, and in viral classification. Staining of nucleic acidsequences could occur in conjunction with or separately from in situamplification of viral RNA sequences.

DEFINITIONS

Certain terminology is used herein as follows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about”, as used herein when referring to a measurable value ismeant to encompass variations of ±20% or ±10%, ±5%, ±1%, or ±0.1% fromthe specified amount, as such variations are appropriate to perform adisclosed method or otherwise carry out the present invention.

The term “adenoviral vector” refers to an adenovirus comprising DNA thatis not normally found in an adenovirus. An “adenoviral vector” can alsobe referred to as a chimeric adenovirus.

As used herein, a prefix to the term “lipoparticle” designates aspecific membrane protein or other specific modification made to thatlipoparticle. For example, “CXCR4-lipoparticle” is defined as alipoparticle comprising the seven-transmembrane receptor CXCR4;“Gag/GFP-lipoparticle” is defined as a lipoparticle comprising a Gag/GFPfusion protein; “Gag/GFP-CCR5-lipoparticle” refers to a lipoparticlecomprising a Gag/GFP fusion protein and the transmembrane receptor CCR5.

A “null-lipoparticle” is defined as a lipoparticle vehicle containing nospecific membrane proteins. As used herein, “no specific membraneprotein” means that a user has not specified a particular membraneprotein to be incorporated into the lipoparticle. “Null-lipoarticles”can still comprise membrane proteins that naturally occur on the surfaceof the cell from which the lipoparticle was produced.

As used herein, the term “LipoProbe” refers to a lipoparticle bearingone or more targeting components and/or one or more signalingcomponents.

As used herein, the term “targeting component” refers to a moleculecomprising one or more target (molecular) recognition domains, and oneor more domains which link it to the lipoparticle.

As used herein, the term “recognition domain” refers to a subunit of thetargeting component which specifically binds to a target molecule.

As used herein, the term “signaling component” refers to a reporterwhich allows an event to be detected or monitored.

As used herein, the term “modifying component” refers to a unit of theLipoProbe, such as an ion channel, that does not in itself possessspecific targeting or reporting characteristics, but which modulates thetargeting and/or reporting ability of the LipoProbe by conferringspecific sensitivities.

As used herein, the term “multimodal detection system” refers to thesimultaneous detection or correlation of one or more reporters.

“Virus,” as the term is used herein, refers to a particle comprising acomplete viral genome and the proteins encoded by that genome in theirnative state.

By the term “applicator” as the term is used herein, refers to anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for attaching a lipoparticle and/or composition of theinvention to a surface, including a sensor surface. Further, theapplicator can be used to contact a ligand and/or a test compound with alipoparticle.

The term “exogenous protein” as used herein, refers to a protein that isnot normally expressed in a cell. An exogenous protein is often, thoughnot exclusively expressed in a cell from a plasmid, a virus, a vector,and the like.

The term “overexpressed” as used herein, refers to a level of proteinexpression that is greater than what is measured in a standard cellline. In some embodiments, a protein is overexpressed at least twotimes, at least three times, at least five times, at least 10 times, atleast 100 times the level of standard cell line. The standard cell linecan be any cell line that expresses the protein of interest. Examples ofstandard cells lines are mammalian cells, mouse cells, human cells suchas, but no limited to HeLa cells, 293 cells, primary cells, stem cells,and the like.

The term, “cellular protein” is used to refer to a protein normallyencoded by the cell and not viral DNA. However, the term also applies toa protein expressed by a recombinant virus wherein a cellular nucleicacid encoding the protein has been inserted into the genome of therecombinant virus for expression therefrom. Furthermore, the term alsoapplies when the protein is provided to a virus or a virus vector in theform of a protein or a peptide.

The term “cellular virus receptor” refers to a membrane protein which iscognate to a viral envelope protein. When displayed on the surface of acell, the cellular virus receptor is capable of binding a cognateenvelope protein and, in some cases, mediating fusion of two lipidbilayers.

The term “cell” refers to any type of living cell. Cells of bothunicellular and multicellular organisms are included. Examples of cellsinclude, but are not limited to, human cells, animal cells, mammaliancells, avian cells, stem cells, primary cells, hybridoma cells,vertebrate cells, invertebrate cells, insect cells, and the like. Asused herein, the term “primary cells” refers to cells that are takenfrom tissues of an organism and are not immortal. Cells can also beimmortalized cells, cancer cells, and the like, and cells that have beenimmortalized. To immortalize a cell is well known to those of skill inthe art.

The term “virus-infected cell” refers to a cell which has been infectedby a virus which comprises a viral protein including, but not limitedto, a viral structural protein in its outer membrane.

The term “producer cell” refers to a cell in which a lipoparticle can begenerated.

As used herein, the term “primary cell” refers to a cell that is derivedfrom a particular tissue and has a limited number of cell divisionsbefore undergoing senescence. Examples of primary cells include, but arenot limited to, keritinocytes, neurons, and the like.

As used herein, the term “cell line” refers to a cell line that is ableto undergo an unlimited number of cell divisions. A “cell line” can alsobe referred to as an immortalized cell line.

As used herein, the term “hybridoma” refers to a type of cell that isboth immortal and capable of producing antibodies. In some embodiments,a hybridoma produces monoclonal antibodies that are of the type IgG,IgA, IgM, and the like.

As used herein, the term “induced cell” refers to a cell that has beentreated with an inducing compound that affects the cell's proteinexpression, gene expression, differentiation status, shape, morphology,viability, and the like. An induced cell can also be referred to as a“modified cell”, a “selected cell,” a “treated cell,” and the like. Insome embodiments an induced cell is contacted with hormones, chemokines,neurotransmitters, and the like.

As used herein, the term “organelle targeting sequence” refers to apeptide sequence that when fused with a second peptide sequence directsthe second peptide sequence to a particular organelle. In someembodiments, an organelle targeting sequence targets a protein to theendoplasmic reticulum or the golgi apparatus.

As used herein, the term “gated” refers to a membrane protein whoseopening and closing is governed by external conditions such as boundproteins or chemicals (e.g. neurotransmitters or hormones), membranepotentials, mechanical means (e.g., vibration or pressure), light, andthe like.

As used herein, the term “non-gated” refers to a membrane protein whosechannel is always open.

As used herein, the term “ionophore” refers to a compound thatfacilitates transmission of an ion across a lipid barrier by combiningwith the ion or by increasing the permeability of the barrier to it.

As used herein, the term “inactivation gate” refers to the part of anion channel that when closed prevents the channel from reopening evenunder conditions that would normally open the channel. The channel canopen once the inactivation gate opens. For example, an ion channel isopened by a change in membrane potential. After a period of time, theinactivation gate closes and prevents the channel from opening againuntil the inactivation gate opens. This is sometimes referred to as the“refractory” period.

As used herein, the term “contaminating protein inhibiting toxin” and“contaminating protein inhibiting ionophore” refers to either a toxin oran ionophore that inhibits the activity of a contaminating membraneprotein (e.g. ion channel). “Contaminating protein” refers to a membraneprotein whose presence is undesirable. The contaminating protein doesnot have to be identified before a contaminating protein inhibitingtoxin or ionophore is used. The “contaminating protein inhibiting toxin”and “contaminating protein inhibiting ionophore” does not inhibit orinterfere with the membrane proteins that are desired.

As used herein, the term “membrane potential” refers to an electricalpotential difference between the intra- and extracellular aqueous phasesof a cell separated by the cell membrane.

As used herein, the term “culturing” refers to the growing, incubating,or propagating of a cell. Examples of culturing include, but are notlimited to, growing, incubating, or propagating cells in suspension, inspinner flasks, in roller bottles, or in a bioreactor.

The term “virus producer cell” refers to a cell in which a virus isproduced. Examples of virus producing cells includes cells that produceadenoviruses, which include but are not limited to chimericadenoviruses.

The term “replication competent” refers to a virus or lipoparticle thatis able to infect or enter into a cell and then replicate and producenew viral particles or lipoparticles. In some embodiments, a replicationcompetent particle comprises the entire viral genome.

The term “infectious” as used herein, refers to a virus or particle thatis capable of both entering a cell and then producing progeny which arecapable of leaving the cell and infecting another cell.

As used herein, a “non-infectious” particle refers to a particle that isnot capable of producing progeny and leaving a cell, but may still beable to enter into the cell.

The term “fusion competent” refers to a viral particle or lipoparticlethat is able to enter a cell. In some embodiments, a lipoparticle can befusion competent without being replication competent.

The term “integration competent” refers to a lipoparticle that comprisesan integrase protein and/or gene that encodes for an integrase.

The term “reverse transcription competent” refers to a lipoparticlecomprising a reverse transcriptase protein and/or gene that encodes fora reverse transcriptase.

The term “protease competent” refers to a lipoparticle comprising afunctional protease gene and/or protein and is capable of cleaving thegag protein.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the lipoparticle and/orcomposition of the invention in the kit for assessing protein binding,identifying ligands for a membrane protein, identifying a compound thataffects a ligand binding with its cognate membrane receptor protein, andthe like, as more fully recited elsewhere herein. Optionally, oralternately, the instructional material may describe one or more methodsof using a lipoparticle of the invention. The instructional material ofthe kit of the invention may, for example, be affixed to a containerwhich contains the lipoparticle and/or composition of the invention orbe shipped together with a container which contains the lipoparticleand/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

As used herein, the term “transfection” refers to the uptake,incorporation, and expression of recombinant DNA by a cell. Methods oftransfection include, but are not limited to chemical transfection,lipid-mediated transfection, electroporation, viral infection, and thelike. Transfection can also be referred to as “transduction” and thelike.

The term “host cell” means a cell that is susceptible to infection by avirus.

The term “protein” refers to peptides and polypeptides.

The term “fragment” in reference to a protein refers to a peptide orpolypeptide comprising at least a portion of a protein. In someembodiments, a fragment comprises at least 5, at least 10, at least 15,at least 20, at least 25, at least 30, at least 40, at least 50, atleast 75, or at least 100 contiguous amino acids of a protein. In someembodiments, a fragment comprises about 5 to about 100, about 5 to about50, or about 5 to about 25 contiguous amino acids of a protein.

The term “competent portion of the genome of a virus” or “competentportion” refers to the portion of the genome of the virus which, whenexpressed in a cell, results in formation of at least one lipoparticle.In some embodiments a competent portion of a genome comprises gag or gagprotein.

The term “antiviral agent” refers to a composition of matter which, whendelivered to a cell, is capable of preventing replication of a virus inthe cell, preventing infection of the cell by a virus, or reversing aphysiological effect of infection of the cell by a virus. Antiviralagents are well known and described in the literature. By way ofexample, AZT (zidovudine, Retrovir® Glaxo Wellcome Inc., ResearchTriangle Park, N.C.) is an antiviral agent which is believed to preventreplication of HIV in human cells.

The term “cytotoxic compound” refers to a composition of matter which,when provided to a cell, is capable of killing the cell.

The term “library” refers to a plurality of nucleic-acid-containingvectors. Said vectors may comprise plasmids, viruses, or othercomponents capable of propagating the nucleic acid. The term “library”may also refer to a large library of chemical or biological compoundsthat are not capable of propagation within a vector. The term “library”can also refer to a phage library or a ribosome display library.

As used herein the term “low-molecular weight organic compound” refersto compounds having a molecular weight less than 3,000. A “low-molecularweight organic compound” can also refer to a compound having a molecularweight less than 1,000.

The term “membrane protein” includes proteins that span the lipidmembrane surrounding a cell, so part of the protein is inside the celland part of the protein is outside the cell. “Membrane protein” can alsoinclude proteins that span a lipid bilayer that is not part of a cell.“Membrane protein” also refers to a membrane spanning protein, amultiple membrane spanning protein, an intracellular membrane protein,an extracellular membrane protein, an organelle membrane protein, andthe like. “Membrane protein” also refers to a protein that is attachedor linked to a membrane, but does not span the membrane. A “membraneprotein” can also be referred to as a “transmembrane protein”.

The term “membrane protein” can also refer to a protein which isexpressed within the lipid bilayer cell surface membrane of a cell. Inthe case of a cellular membrane protein, said protein is encoded by thecell and, at least under certain conditions, is associated with theouter surface of the membrane of the cell. In the case of non-cellularmembrane proteins, the proteins may be derived from a source other thanthe cell expressing the protein, such as a virus, bacteria, yeast, orpathogen. A membrane protein may be a full-length protein, as encoded bya normal cell, or may be a fragment thereof. In some embodiments, themembrane protein is monomeric or multimeric.

A “membrane spanning protein,” as the term is used herein, refers to apolypeptide that spans the cell membrane at least once. That is, thepeptide is typically present in a cell membrane where it spans the lipidbilayer at least once.

As used herein, the term “1-TM” refers to a membrane protein that spansa membrane once. Examples of proteins that can be referred to as “1-TM”include, but are not limited to, CD4, Tva, EGFR, and the like.

“A multiple membrane spanning protein,” as the term is used herein, is apolypeptide that spans the cell membrane at least twice. That is, thepeptide is typically present in a cell membrane where it spans the lipidbilayer at least twice. A multiple membrane spanning protein also refersto peptide that spans the lipid bilayer at least three times, at leastfour times, at least five times, at least six times, at least seventimes, at least eight times, at least nine times, or at least ten times.A multiple membrane spanning protein also refers to peptide that spansthe lipid bilayer three times, four times, five times, six times, seventimes, eight times, nine times, or ten times.

“An intracellular membrane protein,” as the term is used herein refersto a protein that is located inside the cell and is associated with theplasma membrane but does not span it. Examples of intracellular membraneproteins include, but are not limited to farnesylated proteins, lipidmodified proteins, such as Ras, Src, kinases that associate with lipids,such as Protein Kinase C, and PI3-Kinase, and any other protein that isassociated with intracellular side of the plasma membrane. Anintracellular membrane protein can also refer to a membrane proteinlocated on a membrane within the cell, such as, for example, in theendoplasmic reticulum, golgi, nucleus, mitochondria, and the like.

As used herein “associated with the plasma membrane” refers to a proteinthat is either covalently attached to the plasma membrane, but does notspan it, or a protein that interacts through other bonding forces, suchas polar and ionic bonds, with a molecule that is a part of the plasmamembrane.

“Extracellular membrane protein,” as the term is used herein refers to aprotein that is located outside the cell and is associated with theplasma membrane, but does not span the plasma membrane.

As used herein, “an organelle membrane protein” refers to a protein thatis either associated with an organelle membrane or spans the membrane ofan organelle. Examples of organelle membranes include, but not limitedto, golgi membranes and endoplasmic reticulum membranes.

“Exogenous protein” as the term is used herein refers to a protein notnormally found in a specific cell type. For example, a human proteinthat is introduced into a mouse cell.

As used herein, the term “retention signal” refers to a signal thatcauses a compound to be retained at a specific location within the cell.In some embodiments the signal is a peptide or polypeptide. In someembodiments the retention signal retains a protein to the endoplasmicreticulum, nucleus, or golgi apparatus.

The term “non-human animal model of a human disease or disorder” refersto a non-human animal which has been rendered susceptible to infectionby a human pathogenic virus and which, when so infected, exhibits aphysiological condition which is analogous to a symptom exhibited by ahuman infected with the same virus. The term also means a non-humananimal which is susceptible to infection by a non-human pathogenicenveloped virus. When the non-human animal is infected with thenon-human pathogenic enveloped virus, the animal exhibits a pathologywhich is similar to the pathology of a human infected with thecorresponding human pathogenic enveloped virus. By way of example,certain known species of monkeys are susceptible to infection by SIV,giving rise to a disease which is similar to that in humans infectedwith HIV.

The term “pharmaceutically-acceptable carrier” refers to a chemicalcompound or composition with which a lipoparticle of the invention maybe combined for administering to an animal, which in some embodiments isa human. Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, A. Osol, a standard reference textin this field, which is incorporated herein by reference in itsentirety. Preferred examples of such carriers or diluents include, butare not limited to, water, saline, Ringer's solution, dextrose solution,and 5% human serum albumin.

The term ‘biosensor’ refers to an analytical instrument containing abiological sensing element in combination with a convenient readout.

The term ‘biosensor chip’ refers to the surface of a biosensor on whicha test sample is placed in order to detect its biological, chemical, orphysical properties.

The term “ligand” refers to a substance (chemical or protein) that bindsto (matches) a protein. In biosensor applications, the ligand refers tothe molecule attached to the biochip.

The term “ligand,” as used herein, encompasses any protein or compoundthat can bind with a protein present in a lipoparticle. The ligandencompasses a protein or non-protein compound that can bind with aprotein present in a lipoparticle. The term “ligand” can also bereferred to as a “binding partner.” In some embodiments, a “bindingpartner” can also be a monoclonal antibody, a polyclonal antibody, anaffinity-purified polyclonal antibody, a Fab fragment derived from amonoclonal antibody, an immunoglobulin-fusion protein, a single-chainFv, an Fc-fusion protein, peptide, polypeptide, and the like.

As used herein, the term “test ligand” refers to a ligand that is testedto determine if it binds to a lipoparticle comprising a protein. “Testligand” can also refer to a ligand that is tested to determine if itinhibits the binding of another ligand to a lipoparticle comprising aprotein.

As used herein, the term “test sample” refers to a sample that comprisesa ligand, test ligand, and the like. Examples of test samples include,but are not limited to, blood, saliva, serum, cell lysate, organ lysate,tissue homogenate, animal secretions, vaginal secretions, feces, cellculture medium, and the like. In some embodiments, the test sample isdiluted or concentrated, or dissolved with another solvent.

The term “receptor” refers to a protein, often a membrane protein, whichbinds to a ligand of biological significance and transmits theinformation so that it can influence cellular behavior.

A “chimeric virus” is a virus that includes nucleic acid sequences fromtwo different viruses—a primary virus and a secondary virus. The term“chimeric virus” is used to refer to the packaged chimeric virusparticle, while the term “chimeric viral genome” refers to the nucleicacid sequence that is packaged into the chimeric virus. The chimericvirus is capable of transducing a producer host cell and directingproduction of a secondary virus. The components of the chimeric viralgenome include, but are not limited to, the following: (1) primary virusnucleic acid sequences that allow packaging into a primary virusparticle, e.g., packaging signals; (2) optionally, secondary virus genesthat encode proteins for packaging the genome of a secondary virus; (3)optionally, a secondary virus genome; (4) optionally, an expressioncassette with a heterologous gene operably linked to a promoter,typically part of the secondary virus genome. The chimeric viral genomeis typically packaged into a chimeric virus using packaging cells thatcomplement the primary virus nucleic acid sequences. The chimeric viruscan be replication deficient. An example of a chimeric virus includes,but is not limited to, an adenovirus that also has in its genome DNA fora retroviral gag gene.

The term “pseudotype” refers to an enveloped virus that does notcomprise its natural or native envelope protein, which has been replacedby the envelope protein of another virus or another strain of the samevirus.

The term “enveloped virus” refers to a virus comprising an envelopeprotein and a lipid bilayer.

The term “non-enveloped virus” refers to a virus that does not comprisean envelope protein or a lipid bilayer.

As used herein, the term “antigenic composition” refers to compositionthat binds to antibodies. The antigenic composition can be, but does notnecessarily need to be, immunogenic. In some embodiments, an antigeniccomposition can comprise an antibody.

As used herein, the term “immunogenic” refers to a compound orcomposition that is able to generate an immune response including thegeneration of antibodies.

As used herein, the term “generate an immune response” includes humoraland cellular responses.

As used herein, the term “hybridoma” refers to a type of cell that isboth immortal and capable of producing antibodies. In some embodiments,a hybridoma produces monoclonal antibodies that are of the type IgG,IgA, IgM, and the like.

As used herein, the term “induced cell” refers to a cell that has beentreated with an inducing compound that affects the cell's proteinexpression, gene expression, differentiation status, shape, morphology,viability, and the like. An induced cell can also be referred to as a“modified cell”, a “selected cell,” a “treated cell,” and the like.

As used herein, the term “quaternary structure” refers to the way thesubunits fit together. In some embodiments, “quaternary structure”refers to the way polypeptide subunits fit together or form oligomers.In some embodiments the quaternary structure is a homo-oligomer. In someembodiments the quaternary structure is a hetero-oligomer. In someembodiments the quaternary structure comprises a dimer, a trimer, atetramer, and higher-order oligomers.

As used herein, the term “growth property” refers to the division of acell. Examples of defects in growth properties include, but are notlimited to, hyperplasia, neoplasia, metaplasia, cancer, and the like.Examples of cancer, include, but are not limited to breast cancer, coloncancer, lung cancer, skin cancer, brain cancer, leukemia, multiplemyeloma, cervical cancer, uterine cancer, ovarian cancer, prostatecancer, head and neck cancer, bladder cancer, pancreatic cancer, livercancer, and the like.

As used herein, the term “ion-conductance property” refers to a cell'sability to modulate the ion conductance or the ion concentration of thecell. In some embodiments, a defect in ion-conductance is due to adefect in an ion channel protein.

As used herein, the term “signaling property” refers to the cellsability to transmit signals throughout the cell. In some embodiments,the signaling property refers to a signal that originates from amembrane protein and is transmitted inside the cell, the nucleus, orother cytoplasmic compartment (e.g. mitochondria, golgi apparatus, andthe like).

As used herein, the term “mutation” refers to a protein that has atleast one amino acid mutated or changed to another amino acid residue.

As used herein, the term “deletion” refers to a protein that has atleast one amino acid residue removed as compared to the wild-typesequence. In some embodiments at least 2, at least 5, at least 10, atleast 20, at least 50, at least 100 amino acid residues are removed. Theresidues that are removed can also be contiguous.

As used herein, the term “insertion” refers to a protein that has atleast one amino acid residue inserted into the wild-type sequence. Insome embodiments at least 2, at least 5, at least 10, at least 20, atleast 50, at least 100 amino acid residues are inserted. In someembodiments about 1, about 2, about 5, about 10, about 20, about 50,about 100, or about 150 amino acid residues are inserted. In someembodiments about 1 to about 100, about 1 to about 50, about 1 to about30, about 1 to about 10, about 5 to about 10 amino acid residues areinserted. The residues that are inserted can also be contiguous.

As used herein, the term “post-translational modification” refers to amodification of protein that occurs after it is translated from mRNA.Examples of post-translation modification include, but are not limitedto, phosphorylation, dephosphorlyation, sulfation, desulfation,glycosylation, or deglycosylation chimeric modification.

As used herein, the term “chimeric modification” refers to joiningfragments of two proteins to form a chimeric protein. A chimeric proteincan also be referred to as a “fusion protein.” An example of a chimericprotein includes, but is not limited to, a protein that comprises greenfluorescent protein (GFP) and a fragment of another protein. In someembodiments, a fusion protein comprises a linker, a fluorescent protein,a fluorescent peptide, a protease cleavage sequence, a viral protein(e.g. Gag), and the like. In some embodiments, the fusion protein is aGag-fusion protein. In some embodiments a fusion protein is aGag-G-protein fusion protein.

As used herein the phrase “a portion of a lipoparticle's membrane”refers to the lipids and other proteins present in or on the surface ofa lipoparticle. A portion of a lipoparticle's membrane is either theentire membrane of the lipoparticle or less than the entire membrane ofthe lipoparticle.

The “standard cell line” can be any cell line that expresses the proteinof interest. Examples of standard cells lines are mammalian cells, mousecells, human cells such as, but not limited to, HeLa cells, 293 cells,primary cells, stem cells, and the like. In some embodiments, thestandard cell line expresses the protein of interest. In someembodiments, the standard cell line does not express the protein ofinterest or the protein of interest cannot be detected.

As used herein, the term “fluorophore” refers to a compound orcomposition that fluoresces. In some embodiments, a fluorophore is a dyeor a protein.

As used herein, the term “labeling” refers to incorporating afluorophore into a particle or bead. “Labeling” also refers tocontacting a particle with a labeled bead.

As used herein, the term “viral particle” refers to complete virions(viruses), as well as related viral particles, but not single orisolated viral proteins or particles containing a single viral protein.Examples of viral particles include, but are not limited to capsids,core particles, virions depleted of one or more envelope proteins,virion envelopes without the nuclear capsid core, virion envelopefragments and defective or incomplete virions. In some embodiments,viral particles are retroviral particles.

As used herein, the term “Z domain” refers to any Fc-binding domain. Insome embodiments, the Z domain is the Fc-binding domain ofStaphylococcal Protein A, Protein G, or Protein M.

As used herein the term “B1 domain” refers to any Fab-binding domain. Insome embodiments, the B1 domain is the Fab-binding domain ofPeptostreptococcal Protein L.

As used herein the term “target sequence” refers to an oligonucleotidesequence to which another oligonucleotide sequence is able to hybridizeto under varying conditions. In some embodiments, the hybridization isunder stringent conditions. In some embodiments, the target sequence isabout 8, about 10, about 20, about 30, about 40, about 50, about 60,about 0, about 80, about 90, about 100, about 200, about 300, about 400,about 500, about 1,000, about 8 to about 1,000, about 8 to about 50,about 8 to about 30, about 25 to about 100, about 10 to about 100, about100 to about 1,000, about 100 to about 500 nucleotides in length. Insome embodiments, the “target sequence” is specific for a family ofviruses or is specific for a specific pathogen or a family of pathogens.In some embodiments, the target sequence is unique to a specific strainof a virus.

In some embodiments a microfluidic device is narrower than about 1,000microns, about 100 microns, about 10 microns, or about 1 micron.

The present invention also provides methods of hybridizing anoligonucleotide to a target sequence in a lipoparticle, virus, orvirus-like particle. In some embodiments, the method comprisescontacting a oligonucleotide with a lipoparticle, virus, or virus-likeparticle comprising the target sequence under conditions that permithybridization of said oligonucleotide to said target sequence. Thismethod can be used to detect a specific virus, lipoparticle, virus-likeparticle, a virus family, or a type of pathogen.

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein. Those of skill in the art will readily recognize a variety ofnon-critical parameters that could be changed or modified to yieldessentially similar results.

Example 1 Lipoparticle Production Using Core Viral Proteins and MembraneProteins

Murine leukemia virus (MLV)-based lipoparticles were produced by calciumphosphate-mediated transfection of 293T cells in 150-cm2 tissue cultureplates with a plasmid encoding a desired receptor and the pCGP plasmidwhich encodes the MLV gag and pol genes, as performed previously(Hoffman, et al. (2000), Proc. Natl. Acad. Sci. USA, 97:11215-11220).For purposes of optimization, a model GPCR, the chemokine receptor CCR5,was used. The chemokine receptor CXCR4 was also used on occasion. Afterincubation of cells in transfectant overnight, fresh media supplementedwith 10 mM n-butyric acid was added to increase protein expression. 48 hpost-transfection, supernatant was harvested, and cell debris wasremoved by filtering through a 0.45 um filter. The supernatant waspelleted for 60 min in an SW28 rotor at 28,000 RPM through 20% sucrosein PBS, and resuspended overnight in 100 μl PBS. A secondultracentrifugation step through 20% sucrose in PBS was performed, andthe pellet was resuspended in approximately 100 μl of 10 mM Hepes, pH7.0. The lipoparticles were either stored at 4° C. or aliquoted andfrozen at −80° C.

Using 6-well dishes, both the total amount of DNA used (data not shown),and the ratio of receptor to virus core plasmid were systematicallyvaried. Plasmids encoding CCR5 and MLV structural proteins wereco-transfected into 293T cells at ratios ranging from 1:3 to 3:1.Samples were harvested at day 2 and assayed for MLV Gag by ELISA (A405)and CCR5 by dot blot (quantitated by densitometry, in Arbitrary Units).A receptor:gag plasmid ratio of 3:1 produced particles with the highestdensity of membrane protein.

We also varied the length of time that transfection reagent was appliedto cells (4, 6, or 24 hours, data not shown), the time of particleharvest (1-5 days, with media changes or cumulative, data not shown),and the concentration of sodium butyrate applied to cells. Addition ofsodium butyrate (NaB) boosts protein expression by enhancing promoteractivity, but can also result in cellular toxicity. The results indicatethat transfection conditions can make a significant difference not onlyon total particle production, but also on the density of membraneprotein in the particles as measured by the CCR5:Gag protein ratio (datanot shown). Expression of CCR5 was slightly delayed relative to MLV Gag,causing earlier harvests to possess less receptor. NaB enhanced bothparticle production and receptor density per particle, causing anoverall increase in total particle production but an even greaterincrease in CCR5 incorporation.

The results from small-scale experiments were used as a starting pointfor continued optimization at a larger scale. The final protocolincluded transfection of 293T cells by CaPO₄ precipitation in 150 mmdishes, addition of sodium butyrate 24 hours post-transfection, andharvesting supernatant on days 2 and 3.2% fetal bovine serum, ratherthan 10%, was used during production. We also tested the level ofreceptor per virion (CCR5:Gag protein ratio) in the presence and absenceof NaB and at 37° C. and 32° C. As before, NaB had the effect ofincreasing the levels of membrane protein per virion over time. Reducingthe growth temperature to 32° C. heightened this effect significantly,most likely due to slower production of MLV virions but continuedproduction of CCR5 (data not shown).

To achieve high purification, particles must be separated from solubleproteins, cell debris, and membrane blebs released by cells. Previousprotocols loaded supernatant directly into ultracentrifuge rotors, adirect but unscaleable methodology. To address this issue, we adopted analternative purification scheme that is better suited for large-scaleproduction and that is used in the viral vaccine production industry.Briefly, cell debris was removed by low speed centrifugation, andsupernatant was clarified by filtration through a 0.45 μm filter (Step1, Table below). Supernatant was concentrated using tangential flowfiltration (TFF) with a 300,000 MW pore size (Step 2). Viral particlesin the concentrated supernatant were then either exchanged with bufferusing TFF (Step 3a), directly pelleted through a 20% sucrose cushion(Step 3b), or both (Step 4). Our final results (26-fold purificationfrom supernatant), Table 11, compare to results reported for purifiedHIV (84-fold purification from supernatant) (Prior, et al. (1995),BioPharm, 25-35).

TABLE 11 Start Volume Final Volume [Protein] Gag Gag:Protein FoldRelative Stage Purification Stage (ml) (ml) (mg/ml) (A405) RatioConcentrate Purity 1 Unpurified 350 350 0.89 2.91 3.27 1.0 1.00 2Tangential Flow Filt. 350 100 2.14 9.51 4.44 3.5 1.36 3a TFF bufferexchange 175 14 1.33 8.95 6.73 13 2.06 3b Sucrose cushion 175 4.5 0.4532.17 71.5 39 21.9 4 TFF + Sucrose 125 2.0 0.32 27.74 86.7 63 26.5 A405values are multiplied by 10 to adjust for the 10-fold dilution used formeasurement Start Volume indicates the adjusted initial volume afteraliquots and sample divisions have been accounted for

The experiments described above identify conditions needed to optimallyproduce lipoparticles that contain the GPCR CCR5. To test thegeneralization of our methodology to include additional membraneproteins, lipoparticles were prepared by co-transfecting MLV gag/pol(pCGP plasmid) with the receptors indicated (in various eukaryoticexpression vectors), harvesting supernatant two days later, removingcell debris by low speed centrifugation, and dot-blotting for themembrane protein or tags on the protein. Negative controls (media with10% fetal bovine serum, and cells transfected with MLV gag/pol pluspcDNA3 vector) did not demonstrate any reactivity. Multiple antibodieswere required to detect all membrane proteins, so levels ofincorporation cannot be directly compared. The results shown are fromthe same exposure of strip blots. All samples were also assayed for theproduction of MLV structural proteins by ELISA (data not shown). We wereable to detect the membrane proteins CD4, CCR5, IL5Ralpha, DC-SIGN (atype II membrane protein), Shaker (an potassium ion channel), CXCR4,BACE, and Presinilin-1. All proteins appeared to be specificallyincorporated into lipoparticles.

Several controls are worth noting. Sample ‘CCR5 (no MLV)’ included CCR5plasmid but no MLV gag/pol, and the lack of signal rules out artifactssuch as membrane blebs. HA-CXCR4 and CXCR4-HA possess the same HA tagbut on the N-terminus and C-terminus, respectively, indicating that thetag does not interfere with incorporation into lipoparticles.IL5Ralpha-GFP is a single-TM protein with a 27 kDa cytoplasmic GFPfusion, and the signal indicates that proteins with large cytoplasmictails can be incorporated into lipoparticles (data not shown).

Example 2 Production of a Lipoparticle Containing a Human MembraneProtein from a Non-Human Cell

Seven cell types were tested (FIG. 1) for the ability to supportlipoparticle production. Lipoparticles were prepared as described inabove. We also tested dog CF2TH cells (not shown). Since lipoparticleproduction is a function of the number of cells transfected as well asthe health of those cells after transfection, we also tested multipletransfection modalities. Although nearly all cells could be efficientlytransfected by at least one technique (FIG. 1B), few producedlipoparticles efficiently (FIG. 1A), likely reflecting aspecies-specific block in lipoparticle assembly. Murine 3T3 cells(native host for MLV) would likely produce more particles upontransfection optimization. Nevertheless, we identified two cell types,quail QT6 cells and feline CCC cells, that could support moderateamounts of particle production.

Example 3 Production of a Lipoparticle Using a Gag-Fusion Protein

Lipoparticles containing a cellular membrane protein and Gag do notrequire separate proteins to be produced. In some instances, themembrane protein and Gag can be fused together to form one protein. Themembrane protein targets the Gag molecule to the plasma membrane whereGag is able to facilitate the formation of lipoparticles. For example,Gag can be fused to the C-terminus (cytoplasmic) of thesingle-transmembrane protein CD4. When transfected into 293 cells, thisfusion protein will direct the formation of lipoparticles that contain aGag core and CD4 on its exterior. In some embodiments, this fusionprotein will be constructed using a GPCR and Gag. In some instances, theGag fusion protein will be co-transduced into a cell with Gag (unfused)in order to facilitate lipoparticle production.

Example 4 Incorporation of Intracellular Proteins

Traditional methods for drug screening have focused on membrane proteinswhich are present on the extracellular membrane. However, ninety-fivepercent of a cell's membrane structures are located within the cellitself, on organelles such as the mitochondria, golgi, endoplasmicreticulum, and nucleus (Lewin, 1994). Yet direct access to theregulatory proteins on these structures is rarely available because mosthave localization sequences that prevent them from reaching the cellmembrane. Many of these proteins regulate metabolic activities,translocation, transcription, and translation processes that arefundamental to cell biology and disease pathways yet are difficult tostudy.

Like other viral envelope proteins, Hepatitis C(HCV) envelopeglycoproteins E1 and E2 are promising targets for vaccine development.However, E1 and E2 are especially difficult to target with standardimmunization protocols because they form a heterodimer throughnoncovalent interactions and are primarily expressed intracellularly onthe membrane of the endoplasmic reticulum (ER), where HCV capsids areenveloped (9). When expressed in other locations, E1-E2 does not appearto form functional or structurally intact heterodimers. Thus, cell-basedimmunogens are not a viable option for HCV immunization, and HCV virusitself is difficult to grow in large quantities. To form lipoparticleswith E1-E2 complexes, the ER-retention sequences on the cytoplasmictails of E1 and E2 will be removed, allowing the proteins to reach thecell surface and be incorporated into lipoparticles.

In addition to the Golgi and ER, other intracellular organelles alsocontain membrane proteins of interest and are targeted using knownretention signals. For example, peroxisomes are organelles whichparticipate in the breakdown of fatty acids. In humans, proteins aretargeted to the peroxisomes by one of two proteins. Peroxisome signaltype 1 (PTS1) accounts for 95% of the targeting of proteins to theperoxisome. Peroxisomal signal type 2 (PTS2) is a second peroxisomesignal. Similarly, mitochondrial proteins are targeted to themitochondria using a signal sequence which is recognized by a chaperoneprotein called mitochondrial targeting signals (MTS). The mitochondriahas two membranes, the outer membrane and the inner membrane. The outermembrane is very permeable due to a protein called porin. The innermembrane is not permeable due to a protein called cardiolipin. There aretwo transport mechanisms which function to actively move proteinsthrough these membranes. One skilled in the art would recognize thateither PTS1, PTS2, or a mitochondrial signal sequence could be deletedor replaced to retarget peroxisome membrane proteins to the cell surfacefor incorporation into lipoparticles.

An alternative method for generating lipoparticles containingintracellular membrane proteins is to relocalize retroviral Gag so thatit buds from an intracellular location. HIV-1 particle formationnormally takes place on the plasma membrane. However, mutations of thematrix domain of HIV-1 Gag have been shown to retarget the budding tothe Golgi or post-Golgi vesicles (Ono, Orenstein, and Freed 2000). Onecan produce lipoparticles using HIV-1 Gag containing these mutations inorder to capture Golgi membrane proteins from cells onto the surface oflipoparticles.

An alternative method for generating lipoparticles containingintracellular membrane proteins is to use another type of envelopedvirus that naturally buds from an intracellular location. For example,the structural proteins of Hepatitis C (without the E1 and E2 envelopeproteins of HCV) can be used to incorporate ER membrane proteins intothe HCV virion.

Example 5 Use of a Single Chimeric Vector Adenovirus-Gag Vector to MakeLipoparticles

Murine leukemia virus (MLV)-based lipoparticles are produced using anAdenovirus expressing the structural proteins of a retrovirus. AnAdenovirus vector that expresses the MLV gag gene is constructed.Recombinant Adenovirus vectors can be produced using well describedrecombination techniques (Ausubel, et al. (2001), Current Protocols inMolecular Biology). Once the Adenovirus-Gag vector is constructed, it isused to infect HeLa cells at a multiplicity of infection of 10 to ensureexpression of Gag in every cell. One hour post-infection, the cells aretransfected with a plasmid expressing the CCR5 protein from a eukaryoticpromoter (pcDNA3-CCR5). Media is changed after 16 hours and cells areallowed to express the receptor and Gag proteins over the next 2-4 daysat 37° C. Supernatant from these cells is harvested every 8 hours, andlipoparticles from the supernatant are purified as described in Example1.

Example 6 Production Using Two or More Viral Vectors

Murine leukemia virus (MLV)-based lipoparticles were produced using anAdenovirus expressing the structural proteins of a retrovirus. AnAdenovirus vector that expresses the MLV gag gene, in this case fused toa GFP protein, was constructed using Invitrogen's Gateway ViraPower™Adenoviral Expression System. Briefly, the Gag-GFP gene was cloned intoan ‘Entry’ vector that contains lambda integrase recombination sitesthat allow the target to be easily recombined into a variety of other‘Destination’ vectors, in this case an adenovirus recombination plasmid.The adenovirus destination plasmid was transfected into HEK-293 cells,and cultured until recombinant adenovirus emerged. Recombinantadenovirus was checked by Western blot for Gag expression using anti-Gagrabbit sera. Separately, another Gag-GFP recombination Adenovirus wasconstructed using pCMV-LINK vector recombination with purifiedAdenovirus DNA, as instructed (Current Protocols in Human Genetics,1995). Recombinant Adenovirus vectors can also be produced using otherwell described techniques or kits (Ausubel, et al. (2001), CurrentProtocols in Molecular Biology). Once the Adenovirus-Gag vector wasconstructed, it was used to infect 293T, BHK, HeLa, and NIH-3T3 cells ata multiplicity of infection of 10-500. Simultaneously, an Adenovirusexpressing the membrane protein CCR5, produced using pCMV-LINK, was alsoused for infection of the same cells. Media was changed after 16 hoursand cells were allowed to express the receptor and Gag proteins over thenext 4 days at 37° C. Supernatant from these cells were harvested every8 hours. One skilled in the art would recognize that similar viralexpression systems could also be constructed using other viral vectors,including baculovirus, vaccinia, semliki forest virus, sindbis virus, orother alphaviruses, and expression in appropriate cell types.

Example 7 Production Using Vaccinia Virus

Recombinant vaccinia vectors will be produced by cloning MLV Gag andCXCR4 into the plasmid pSC60. Flanking the gene in this vector arerecombination sites that allow the gene to be recombined into a wildtype vaccinia virus (WR) when plasmid and virus are introduced into thesame cell. The recombination event results in loss of the thymidinekinase gene within the virus, from which the virus can be selected (BrdUresistance). Once recombined, the recombinant virus is very stable andcan be grown to high titers because the virus is replication competent.Once expanded, the vaccinia will be used for the production oflipoparticles. Lipoparticles and cells expressing lipoparticles will becharacterized by Western blot for Gag expression using anti-Gag rabbitsera and for CXCR4 expression using an anti-epitope (V5) antibody.Although vaccinia can kill infected cells within 2-3 days, this lengthof time can be prolonged by addition of chemicals that inhibit vacciniareplication (AraC and rifampicin).

Example 8 Production Using Vaccinia Virus Encoded T7-Polymerase

Murine leukemia virus (MLV)-based lipoparticles are produced using aninfection-transfection system utilizing vaccinia-expressedT7-polymerase. HEK-293T cells are transfected with a plasmid expressingthe CCR5 protein from a T7 promoter. The T7 promoter is not normallyactive in eukaryotic cells, but can be turned on by expression of the T7polymerase protein within the same cell. Three hours post-transfection,the same cells are infected with a vaccinia expressing the T7 polymerase(vTF1.1, MVA-T7-polymerase, or vTF7-3). Media is changed after 16 hoursand cells are allowed to express the receptor and Gag proteins over thenext 1-4 days at 37° C. Supernatant from these cells is harvested every8 hours, and lipoparticles from the supernatant are purified asdescribed in Example 1.

Example 9 Production Using Semliki Forest Virus

To create replication-competent SFV replicons, we will construct aplasmid containing an SFV replicon that is expressed from a CMV promoterand results in the production of SFV RNAs capable of replication in thecytoplasm of transduced cells. The precise 3′ terminus of the SFVreplicon RNA will be generated by ribozyme-mediated cleavage, a strategyused for similar constructs (West Nile Virus, Dengue virus, and SFVreplicons) using the identical ribozyme (Khromykh, et al. (1998), JVirol, 72:5967-77, Khromykh, et al. (1997), J Virol, 71:1497-505, Pang,et al. (2001), BMC Microbiol, 1:18, Shi, et al. (2002), Virology,296:219-33, Varnavski, et al. (1999), Virology, 255:366-75). SFV vectorsexpressing MLV gag and CXCR4 will be constructed. To produce SFV viralvectors, HEK-293T cells will be co-transfected with the plasmid encodingthe SFV replicon and plasmids encoding the structural genes of SFV(capsid, E1 and E2/3). SFV will be harvested in the supernatant of thesecells and stored frozen until used for infection of BHK cells toinitiate lipoparticle production. BHK cells infected by recombinant SFVvectors will be characterized by Western blot for Gag expression usinganti-Gag rabbit sera and for CXCR4 expression using an anti-epitope (V5)antibody.

Example 10 Capture of Naturally Expressed Membrane Proteins on the CellSurface

The previous examples have focused on generating lipoparticlescontaining specific membrane proteins deliberately engineered into thelipoparticle. Lipoparticles can also be produced according to theinvention from cells which contain native membrane proteins of interest(e.g. primary cells, stem cells, cell lines with interesting propertiesor receptors). Lipoparticles are produced using an Ad-Gag vector todeliver the structural protein of MLV. In the present example, however,no membrane protein will be exogenously introduced—the membrane proteinwill be expressed naturally from the cells chosen for production. Thiswill allow us to capture naturally expressed membrane proteins fromdesired cell types.

Neural stem cells are cells which have the ability to self-renew, andcan divide to produce all the cell types of the nervous system. Onedifficulty in neural stem cell biology has been the identification ofthese cells. The protein Nestin has been proposed to be a marker ofthese cells, yet Nestin is also expressed in other cell types such asastrocytes. At best, a sub-population of Nestin positive cells can actas neural stem cells. Similar problems exist with other neural stem cellmarkers, such as Musashi.

In order to generate lipoparticles from neural stem cells, flowcytometry is used to obtain a population of Nestin positive cells fromprimary culture. Lipoparticles from the Nestin positive cell membranesare then produced by infecting Nestin positive cells with an adenovirusthat expresses MLV Gag. As the Gag drives budding from the cells,lipoparticles are formed containing the membrane proteins of the Nestinpositive cells.

Example 11 Identification of an Infectious Pathogen

A biosensor chip is created with 95 lipoparticles in positions 1-95 andan empty position at position 96. The 95 lipoparticles each have adifferent membrane protein that bind to a different infectious agent.Among the 95 are membrane proteins directed against Ebola, anthrax,cholera, plague, E. Coli., chicken pox, HIV, HSV, salmonella receptor,Hepatitis A, Hepatitis B, Hepatitis C, and Influenza, including themembrane proteins CCR5, CXCR4, CD-SIGN, CD-SIGNR, CFTR, CD44 (shigellareceptor), mannose receptor MRC1 (ricin toxin), alpha dystroglycan andbeta dystroglycan (Lassa Fever and LCMV receptors), MCAT-1 (murineleukemia virus) and the anthrax toxin receptor. The lipoparticles areattached to the biosensor. A serum sample is screened against the arrayof lipoparticles. The biosensor indicates that the individual isinfected with anthrax.

Additional membrane proteins, including all membrane proteins known tobe receptors for pathogens and toxins, can be included.

Example 12 Using the Lipoparticle Biosensor to Identify OrphanReceptors, Orphan Ligands and Drug Targets

Bacterial and viral pathogens enter their host cells (and eventuallykill or weaken them) using a cellular receptor. Many of these receptorshave been identified, but many have not. A lipoparticle biosensor isused to identify membrane proteins that interact with known pathogens.For example it is unknown how the SARS virus interacts with the cellsurface to infect the cell. Screening a lipoparticle biosensorcomprising the membrane proteins of a cell with a sample containing SARSvirus will enable the identification of the membrane proteins which itinteracts with. This screening will take place similar to the screeningof the infectious agents in Example 11. Knowledge of these membraneproteins may better direct the development of therapeutics to combatSARS infection.

One skilled in the art would recognize that any virus, bacteria, toxin,etc., with an unknown cell receptor can be screened to discover unknownbinding partners on the cell membrane. This will prove useful for theidentification of infectious pathogens because less than 25% of them arelinked to identified receptors. Similarly, one skilled in the art willrecognize that the lipoparticle biosensor can be used to identify thetarget of a drug, protein, or peptide with an unknown surface proteininteraction. Conversely, where known membrane proteins exist on theCSRB, soluble partners could be sought by washing large numbers ofsamples over the array.

Biosensors are capable of detecting ligands in complex mixtures atconcentrations well below their K_(D), and ligand binding can bemonitored in real time, permitting accumulation of ligand duringcontinuous flow across the surface. As such, a promising application forthe lipoparticle biosensor is the identification of ligands in complexmixtures, often for “orphan” receptors that do not have known ligands(“ligand fishing”) (Nedelkov, et al. (2001), Biosensors &Bioelectronics, 16:1071-1078, Nedelkov, et al. (2002), Proteomics,2:441-446, Williams (2000), Current Opinion in Biotechnology, 11:42-46).Ligand fishing is performed on supernatant from the FC36.12 cell line tosearch for 13-chemokines. Cell supernatant fractions derived from theFC36.12 CD8⁺ T cell line were used in 1995 to identify the chemokinesRANTES, MIP1α, and MIP1β as inhibitory factors of HIV (Cocchi, et al.(1995), Science, 270:1811-1815). Supernatant is flowed across alipoparticle biosensor containing CCR5, and binding to CCR5 is monitoredin real time. To verify that binding is due to β chemokines,non-competitive antibodies against the β-chemokines are flowed across todetect their presence on the lipoparticle biosensor. Alternatively,ligand is eluted (removed) from the chip using a regeneration solution(high salt, low pH, or high pH buffer) and assayed by Western blot.Additional controls can include non-specific lipoparticles, supernatantfrom other cell types, and inhibition of binding with blocking agents(CCR5 inhibitors, MAbs). In some embodiments, concentrated supernatantsare used.

Example 13 Using the Lipoparticle Biosensor for De Novo Ligand Design

A large number of membrane proteins remain orphan receptors, withoutidentified natural ligands. Other membrane proteins, such as CXCR4, arelinked to identified ligands, but the ligands have undesirablecharacteristics (low affinity, low specificity, labile, highly charged).In such cases, de novo design of a better ligand, often starting withrandom peptides, is highly desirable. Even for proteins with wellfunctioning ligands, de novo ligand design using peptides is often thefirst step in the process of rationally designing small moleculeinhibitors.

A phage library displaying random 7-mer peptides is used to pan aCXCR4-biosensor chip to perform de novo ligand design. The “Ph.D. PhageLibrary” (New England BioLabs) contains over 2×10⁹ independent clones,each carrying a random 7-mer peptide that in sum represents everypossible permutation of seven amino acids. Phage are flowed over thelipoparticle biosensor surface, washed, and then eluted in successivelymore stringent (lower pH) elutions to collect pools of increasinglyselective peptides. The pools with highest affinity for the target areamplified and re-screened to select phage with the highest affinity (oravidity in this case) for the target receptor. Approximately twentyhigh-affinity phage from each stage of screening are sequenced todetermine any consensus sequence. One skilled in the art would recognizethat phage panning could also be conducted using whole virus orvirus-like particles.

Example 14 Using the Lipoparticle Biosensor to Generate ProteinInteraction Maps

The lipoparticle biosensor is used to quantify the reactivity of asecreted protein with the complete set of membrane proteins that it iscapable of binding to. Similar interaction maps have been deciphered forthe yeast proteome and for subsets of human cytoplasmic networks, butnot for membrane proteins. Biosensors are capable of detectingaffinities of interaction from sub-picomolar to micromolar, enabling thecomplete range of reactivities to be characterized. The chemokinesMIP1α, MIP1β, and RANTES are assayed on the cell surface biosensor todetermine the complement of receptors to which they bind and thekinetics of interaction with each membrane protein. This screening ofthe lipoparticle biosensor will take place as described herein.

Example 15 Using the Lipoparticle Biosensor to Determine the Toxicity ofReactivity of a Drug

Lead drug candidates are tested on the lipoparticle biosensor forbinding to membrane proteins that could cause unwanted biologicaleffects (a component of standard toxicity testing). Currently, onlyclosely related targets are tested, and side effects that could havebeen avoided are often identified late in drug development. Thisscreening of lead drug candidates will take place similar to asdescribed herein. Briefly, each drug candidate is diluted and washedover the lipoparticle biosensor. The receptor biosensor will detectwhere the drug candidate interacts with a membrane protein. Particularattention will be paid to interactions with membrane proteins on thearray which are known to be linked to negative drug side effects.

Known drugs can also be screened on the lipoparticle biosensor todetermine if they interact with other receptors. For example, samplescontaining Cimetidine (Tagamet™) are screened on the lipoparticlebiosensor. Cimetidine is known to interact with the Histamine H2G-protein coupled receptor, which plays a role in ulcers. It is notknown, however, how strongly Cimetidine interacts with many othermembrane proteins, a question that can be answered using thelipoparticle biosensor. For example, the ability of drug such asCimetidine to bind selectively to the Histamine H2 GPCR and not theHistamine H1 or H3 GPCRs is a strong determinant of effectiveness andside-effects. This screening of the lipoparticle biosensor will takeplace as described in Example 11.

Example 16 Using Lipoparticle Biosensor to Generate a Profile ofCompounds Sharing a Measurable Characteristic

The CSRB will be used to develop a profile of compounds sharing anymeasurable characteristic. Measurable characteristics could include, butare not limited to, toxicity, efficacy, solubility, absorption profiles,or the ability to cross the blood-brain barrier. The lipoparticlebiosensor is able to identify similarities in the binding to cellsurface proteins of compounds which share characteristics. Conversely,the lipoparticle biosensor can identify differences in binding ofcompounds which have different characteristics.

For example, a battery of compounds which have been determined to benon-toxic (or “safe”) are screened against the lipoparticle biosensor.Each of these safe compounds (known drugs with non-toxic profiles) isscreened on the lipoparticle biosensor with techniques similar to thescreening described in Example 11. Membrane proteins on the lipoparticlebiosensor which interact with all of the safe compounds will beconsidered “hits” and will constitute a positive profile of a safecompound. Conversely, membrane proteins on the lipoparticle biosensorwhich interact weakly or rarely with the safe compounds will beconsidered “misses.” A similar screen using toxic compounds willgenerate “hits” and “misses” for toxicity. By combining the safe “hits”(membrane proteins that are bound by compounds with good safetyprofiles) with the toxic “misses” (membrane proteins that are bound bycompounds with poor safety profiles) a profile of safe membrane proteinsare generated. Such information is then used to predict thecharacteristics of other compounds.

One skilled in the art will recognize that a similar system of “hits”and “misses” could be used to generate profiles of any group ofcompounds which share a similar measurable characteristic. Compoundswhich elicit similar side effects, beneficial outcomes, or druginteractions could be screened with the lipoparticle biosensor todetermine a profile of that characteristic.

Example 17 Using the Lipoparticle Biosensor to Pre-Screen for Non-ToxicMolecules and Compile the “Safe Candidate” Library

Chemical libraries are available from commercial sources for assayvalidation. The library is chosen to contain known receptor agonists andantagonists, non-specific analogs within the same chemical family, andother non-specific compounds. This library is used to screen the cellsurface biosensor for “lead compounds” in a trial drug screen. Drugcandidates matching the profile of a safe drug (binding to membraneproteins on the lipoparticle biosensor (see Example 16) which have beenassociated with known safe drugs) are added to a Safe Candidate library.The Safe Candidate library will serve as a basis for traditional drugdiscovery with the advantage being that the candidates are pre-screenedto match a profile for non-toxicity.

In this way a large library can be built even before specific targetsare linked to specific diseases or phenotypes. For example, abiotechnology company may discover that a new gene (X) is involved incancer. Rather than screen for small molecule compounds or antibodieswhich may turn out to be toxic, the company could screen the SafeCandidate Library, with higher confidence that any drug identified willbe not toxic. In this manner, the early stages of drug development canbe hastened and better molecules for human application (e.g. toxicity,bioavailability) can enter drug discovery.

One skilled in the art will recognize that candidate libraries can beconstructed such that many characteristics of candidate compounds can bepredicted based on profiles of known drugs. Ideally, a library ofcandidate molecules which fit the profile of non-toxic, cheap, easilydelivered drugs will be generated.

Example 18 Using the Lipoparticle Biosensor to Identify the Interactionsof Membrane Proteins with Candidate Compounds

Because the lipoparticle biosensor can express all membrane proteins,small molecules can be screened against these proteins to identifyreactions. In this manner, purified monoclonal antibodies andsmall-molecules (e.g. organic drugs) can be identified that targetproteins of specific structures or phenotypes of defined function, evenwithout knowing the precise target of interest.

A random, purified monoclonal antibody from a defined hybridoma clone isused to screen the lipoparticle biosensor. The same could be done for achemically pure small-molecule. The protein that the antibody reactswith can then be defined. Monoclonal antibodies should reactspecifically with only one protein. This may be useful if antibodies (orsmall molecules) have effects of interest, but their targets are notknown. Moreover, if a random antibody or chemical binds to a smallnumber of targets on the array (ideally a single target), then thespecificity of that antibody or chemical is defined. Screening a largenumber of purified monoclonal antibodies or chemically puresmall-molecules will enable the development of a library ofantibodies/chemicals that have already been pre-screened for thespecificity desired. Moreover, if these antibodies or chemicals are alsopre-screened for their toxicity, bioavailability, etc., a library ofcompounds will arise that has known specificity and that arepre-screened to be better compounds for drug development—i.e. a libraryof lead compounds to defined targets. Targets of complex nature (e.g.membrane receptors, glycosylated proteins) are particularly amenable tothe array. One skilled in the art will recognize that any protein,compound, antibody, or natural or synthetic drug could be screened usingthe lipoparticle biosensor in this manor.

Example 19 Using the Lipoparticle Biosensor to Investigate Changes inMembrane Protein Ligands During Disease Progression or in DifferentDisease States

The multiplexed nature of the lipoparticle biosensor enables not justsingle binding events to be measured, but patterns of reactivity. Likegene chips, where every feature of the array may not be completelyknown, correlates of reactivity can be as predictive (or more) ofdisease than understanding single pathways of interaction. Complexsamples, such as serum, urine, and saliva from individuals with avariety of disorders, are run over the cell surface biosensor tocorrelate patterns of reactivity with the individual's health. Theapplication of these samples will proceed as described in Example 11.Multifactorial causes of disease can be identified even if multiple newligands combine to cause a physiological effect. The serum of anindividual will be tested throughout the course of a viral or bacterialinfection to correlate cytokine immune response with diseasepathogenesis and drug treatment. A lipoparticle biosensor can measureand quantitate dynamic changes of chemokines, hormones, and biologicallyactive peptides simultaneously.

In this way a profile of the disease will be generated similar to thegeneration of a profile of a drug candidate described above. Forexample, blood samples taken from an HSV2 patient during the latentphase of the disease may bind to a different, yet predictable, set ofmembrane proteins than samples taken during the active phase. Thisinformation will serve multiple purposes. First, it will help to definedisease state based on measurements other than clinical assessment, andsecond and more importantly, it will identify membrane proteinscorrelated to changes in the disease. These membrane proteins arepotentially important research targets for understanding the underlyingdisease state and for the potential development of novel treatments.

Example 20 Using the Lipoparticle Biosensor to Investigate Differencesin Membrane Proteins Expressed in Different Samples of PrimaryTissue/Cell Types/Organs

The lipoparticle biosensor will be used in this example to generate aprofile of different sources of ligands. Specific cell types, tissues ororgans are obtained, lysed to liberate the proteins, and screened usingthe lipoparticle biosensor. In this way, all of the membrane proteinsinteracting with proteins from a specific sample are identified, anddifferences between sources will be obtained.

Two kinds of tumor samples are obtained, malignant and benign. Thesetumors are lysed by mechanical homogenization and the soluble proteinsisolated by eliminating the membranes using centrifugation. Themalignant tumor samples are examined using the lipoparticle biosensorusing a similar protocol to the one described in Example 11. In thismanor the cell membrane receptors that bind to the soluble proteins inthe malignant tumor will be identified. Likewise, the benign tumorsample will be examined using the lipoparticle biosensor. Differences inreceptor binding will be evident by comparing the samples.

Example 21 Receptor Biosensors Designed to Represent Two or MoreDifferent Populations

Lipoparticles are generated from various cell types, tissues, or organs.An organ-specific receptor biosensor is constructed by makinglipoparticles from all of the human organs and arraying them onto abiosensor. Lipoparticles are generated as described above, except thecell source will be primary tissue which is not expressing membraneproteins other than those found naturally on the cells. Candidatelibraries could be screened for candidates that interact with a specificorgan but not with others. Similar receptor biosensors could beconstructed to investigate specific binding in tissues or specific celltypes. Such a biosensor could also be constructed to look at differencesbetween tumor kinds, healthy vs. diseased organs or tissues, ordifferences before and after stimulation of a region using a chemical orother stimulus. One skilled in the art will recognize that anypopulation with distinct differences in membrane proteins could becompared in this way.

Example 22 Receptor Biosensors Designed to Represent Two or More Species

Receptor biosensors are designed such that the lipoparticles containingmembrane proteins from human and bacteria are included on the samebiosensor. The lipoparticles will be produced as described above withthe exception being that the membrane proteins expressed on the cellsurface are bacteria. The human receptor lipoparticles will compriseproteins only found in humans. Such a receptor biosensor is screenedwith a library of potential antibiotics to find candidates whichinteract with bacterial membrane proteins but do not interact with humanmembrane proteins.

It is evident to one skilled in the art that other lipoparticlebiosensors containing two or more samples from different sources oflipoparticles could be constructed to distinguish differential candidateinteractions.

Example 23 Portable In-Field Diagnostics

A lipoparticle biosensor surface is used to create in-field use devicesfor detecting samples interactions with membrane proteins. This toolcomprises a receptor biosensor surface including relevant proteins, aportable biosensor, and a microprocessor to interpret the data andprovide real time readout.

Versions of this tool will be designed using a lipoparticle biosensor,which can test for infectious agents, water quality, food quality, andthe like.

The samples analyzed by the lipoparticle biosensor kits can originatefrom many sources, including but not limited to biological fluids takenfrom an organism, liquid samples from nature, man made liquid samples,aerosols, or dissolved biologic or man-made solids from various sources.Size and weight of the receptor biosensor are not essential to theinvention, however the preferred size will be a biosensor that can becarried in one hand, weighing not more than fifty pounds, morepreferably not more than ten pounds, and more preferably not more thanone pound.

Example 24 Lyophilization of Lipoparticles

To test their stability, lipoparticles starting in 10 mM Hepes 7.0, 30mM NaCl were suspended in increasing amounts of sucrose, trehalose, orglycerol, and lyophilized overnight. The lyophilized mixture wasresuspended in water to their original starting particle count of 20 E+6per ul, based on the pre-lyophilization counts. Once resuspended, thelipoparticles were tested for retention of membrane protein structure byVELISA using a CXCR4-specific conformation-dependent monoclonalantibody. The results demonstrated that the addition of sucrose,trehalose, or glycerol allowed the retention of membrane proteinstructure during lyophilization, as seen by the continued binding ofCXCR4 to the MAb 447.08 (FIG. 4A). The results also demonstrate that thelipoparticles lose their native structure without such additives, asdemonstrated by the lack of binding of lipoparticles lyophilized withoutadditive (0%). Controls included in the same experiment indicated theadditives alone did not produce these effects (FIG. 4B).

The lipoparticles were also tested for size and purity afterlyophilization by dynamic light scattering, showing a monomodal 200 nmpeak when lyophilization occurred in the presence of sucrose, similar tothe type of peak seen with unlyophilized lipoparticles (FIG. 5). Withoutsucrose, the lipoparticles showed spurious peaks at higher sizes,suggesting aggregation or destruction of the lipoparticles. A similarexperiment was also conducted by drying the lipoparticles at roomtemperature and testing for the retention of CXCR4 structure bybiosensor analysis. These lipoparticle also retained CXCR4 structure.One skilled in the art would recognize that other drying techniques andother additives such as glucose could also be used.

Example 25 Attachment to Biosensor Surfaces

Lipoparticles were immobilized to a BIACORE C1 chip through twonon-covalent attachment methods. In the first, 400 RU of WGA (wheat germagglutinin, 1.0 mg/ml in Hepes pH 7.0, 100 mM NaCl) was covalentlyattached via amine coupling to the C1 chip in flow cell 4. 6000 RU ofNeutrAvidin (Pierce, 1.0 mg/ml in Hepes pH 7.0, 100 mM NaCl) wasimmobilized in flow cell 2 by the same reaction. Both proteins werediluted to 0.5 mg/ml immediately prior to injection in 10 mM pH 4.5acetate buffer. Injections were performed at 20 .mu.l/min and wereproceeded by 7 minute injections of 5× (1M and 0.25M) EDC/NHS solutions,and proceeded by quenching of the activated carboxylates with a 7 minuteinjection of 1M ethanolamine.

Lipoparticles were then injected at a flow rate of 511/min.Lipoparticles diluted to 30 million particles per microliter in Hepes pH7.0, 100 mM NaCl were injected over the attached WGA in flow cell 4 for720 seconds, resulting in approximately 3000 RU of immobilized particles(FIG. 6).

Biotinylated lipoparticles were prepared with Sulfo-NHS-LC-biotin,performed for one hour in pH 8.0 PBS buffer with an excess of reagentrelative to total protein content of 1000 to 1. The chemicallybiotinylated particles were in 10 mM Hepes, pH 7.0 100 mM NaCl, and werepurified after biotinylation by column chromatography on anionicexchange resin, and further purified by high speed centrifugation andbuffer exchange. Chemically biotinylated particles, determined to be 10million particles per microliter, were injected over flow cell 2containing attached neutravidin at a rate of 5 microliters/minute for 10minutes, resulting in approximately 1100 RU attachment (FIG. 6).

One skilled in the art would recognize that lipoparticles, viruses, orvirus-like particles could be captured in similar ways. Lipoparticles,viruses, or virus-like particles could also be captured to surfaces bymixing the particles in solution with a capture agent, such asWGA-biotin, and then flowing the particles over a suitable surface, suchas avidin. Lipoparticles could also be captured using a membrane proteinin the lipoparticle, such as a transmembrane-anchored avidin fusionprotein or a fusion protein containing a His-tag, that allows attachmentof the lipoparticle to a suitable surface such as biotin or Ni+2.

Example 26 Attachment to a Hydrophobic Biosensor Surface

An “E1” chip was created by incubation of gold chip (BIACORE Au Sia kit,cleaned by rinsing with 100% ethanol) in 0.1 mM DSPE-PEG(2000)-PDP(90/10, v/v EtOH, diH.sub.2O), a gold (Au) reactive PEG moleculefunctionalized with lipid tail groups that acts as attachment points forlipoparticles. The surface was rinsed in 100% EtOH, followed bydiH.sub.2O, dried with pressurized air, and assembled into a BIACOREchip cartridge as per kit directions. The chip was prepped with threeinjections of 0.2% deoxycholate prior to injection of lipoparticles.Lipoparticles (diluted with Hepes pH 7.0, 100 mM NaCl to a finalconcentration of 30 million/ul) were injected for 10 minutes at 5ul/min, resulting in approximately 3200 RU of binding (FIG. 7).

Example 27 Making Lipoparticle Biosensors Based on Lipoparticles Arrayedon Slides

The spotting of lipoparticles onto slides offers the fastest method forlarge-scale multiplexing of lipoparticles on a biosensor. Biosensorsurfaces are currently being developed as chips for arrays, in additionto more traditional 96- and 384-wells. The ability to arraylipoparticles on a biosensor chip would enable the detection ofthousands of membrane protein interactions simultaneously. When largesets of binding interactions are measured simultaneously, patterns ofbinding can be indicative of disease and health profiles. Lipoparticlescan be disrupted by harsh conditions and the proteins within them aredependent on the integrity of the lipid membrane for retention ofstructure. Dehydration while arraying may cause a significant loss offunctional activity.

Two procedures are used in the spotting of lipoparticles onto a glassslide. First, one can use a surface chemistry (gamma-aminopropylsilane,GAPS) that has been reported to stabilize membrane proteins within lipidenvironments on the surface of slides (Fang, et al. (2002), J Am ChemSoc, 124:2394-2395). This chemistry was used by this group to attachmembrane vesicles to glass slides, and the membrane proteins weredemonstrated to adhere, withstand extensive washing, and maintain theirability to bind ligands. Second, one can include preservatives to thelipoparticles preparation that can stabilize membrane structures evenunder extreme conditions. For Example, the simple carbohydrate trehalose(naturally found in insects and plants to help them withstand harshconditions) has proven to stabilize viruses, proteins, and lipids underconditions including lyophilization, dehydration, and heating(Bieganski, et al. (1998), Biotechnol Prog, 14:615-620, Paiva, et al.(1996), Biotechnol Annu Rev, 2:293-314). Other additives (glycerol,sucrose, gelatin) have also been used as preservatives.

One skilled in the art will recognize that while the preferred use oflipoparticles linked to a glass slide will be screening by biosensor, itis also possible to screen the slide by labeling the probes. The glassslide biosensor will be hybridized with marked probes. The probesconsist of any sample to be tested for interaction with membraneproteins which have been labeled using fluorescent molecules such as Cy3or Cy5, radioactive molecules, enzyme-linked molecules, or biotinylatedmolecules. Labeled slides can be read using a variety of methodsdepending on the labeling technique, including using computerizedreaders currently produced to read microarrays. One skilled in the artwould recognize that such probes can consist of a fluorescent,enzymatic, biotinylated, or radioactive tag.

Example 28 Lipoparticles and Biosensors to Detect Multi-ProteinComplexes

The propensity of multiple proteins to form larger complexes is afundamental attribute of pathways going to, from, and within a cell(also sometimes termed “proteomics”). Protein interaction maps have beenconstructed using the biosensor (Nedelkov, et al. (2001), Biosensors &Bioelectronics, 16:1071-1078, Nedelkov, et al. (2001), Proteomics,1:1441-1446) and other techniques, but such maps are difficult toconstruct for membrane proteins. Lipoparticles containing the chemokinereceptor CXCR4 (or CCR5) are attached to a biosensor chip. A complex ofHIV gp120 and sCD4 (soluble versions of each that lack transmembranedomains) are bound to the CXCR4 molecule, as previously performed(Hoffman, et al. (2000), Proc. Natl. Acad. Sci. USA, 97:11215-11220).Non-blocking antibodies to gp120 and sCD4 are then sequentiallyintroduced. Next, secondary antibodies against the primary antibodies(e.g. anti-rabbit, anti-mouse) are sequentially introduced. Finally,antibodies targeted to tags on the secondary antibodies (horseradishperoxidase, alkaline phosphatase) are introduced. The detection of theseproteins indicates that multi-protein complexes can be detected.

Example 29 Lipoparticles and Biosensors for Antibody Characterization

One of many applications for the lipoparticle-biosensor is thecharacterization of antibodies directed to membrane proteins. There aremany sources of large panels of candidate therapeutic monoclonalantibodies against membrane protein targets that need to becharacterized for kinetics and specificity. Traditional methods ofanalysis for membrane proteins (competitive radioligand binding orELISA) are often not sufficient to derive the information (on-rate,off-rate, affinity, specificity) that they desire in an efficientmanner. For example, a panel of 10-20 CXCR4 and CCR5 antibodies werepreviously isolated and characterized (Baribaud, et al. (2001), J.Virol., 75:8957-8967, Lee, et al. (1999), J. Biol. Chem.,274:9617-9626). The lipoparticle biosensor is to be used to measure eachantibody's on-rate, off-rate, affinity, and specificity. For eachantibody, these measurements can be gathered in a single experiment.Monovalent Fab fragments of each MAb are used to ensure accurateanalysis of kinetic data.

In another embodiment, the lipoparticle biosensor compriseslipoparticles that express membrane-bound antibodies. Lipoparticles areprepared that contain membrane-bound antibodies. The antibody can be awhole antibody or a fragment of an antibody such as a Fab fragment, animmunoglobulin-fusion protein, a single-chain Fv, an Fc-fusion protein,or combinations thereof. Each antibody can be specific for a known orunknown epitope or target proteins. The lipoparticles are spotted in anarray on the lipoparticle biosensor. The array is screened againstproteins of interest in order to identify which, if any, of theantibodies on the array bind the protein of interest. The ligand used tobind the array of lipoparticles can be an antibody, protein, peptide,drug, or another lipoparticle. In another embodiment, the array isprepared using lipoparticles expressing T-cell receptors.

Example 30 Lipoparticles and Biosensors May be Used to Track the Qualityof an Immunization or to Detect Antibodies Against Particular Infections

Blood samples are taken at various stages after an immunization. Theantigen which is the target of the immunization is included on thelipoparticles. The addition of boosters will depend on the binding tothat antigen on the biosensor. Upon measuring low binding, a booster isadministered to the individual or animal. Upon measuring high binding,no booster is administered.

This technology is similarly used to determine if a patient has beenexposed to infections. Lipoparticles on the array contain antigensrecognizable to antibodies circulating in the blood of the patients.Epitopes for diseases such as influenza virus, respiratory syncytialvirus, HSV1, HSV2, varicella zoster, Epstine Bar Virus, HHV8, ordifferent kinds of HPV are included on the array. One skilled in the artwill recognize that any protein that is potentially recognized byantibodies generated against a disease can be included. In someembodiments, this lipoparticle biosensor is used for diagnosis ofinfections, giving the physician the ability to screen for hundreds ofpotential infections simultaneously.

Example 31 Kinetic Analysis of MAbs Directed to Membrane Proteins

As a result of optimization, we can reproducibly attach lipoparticles toa BIACORE biosensor chip, detect the binding of unlabeled molecules assmall as 8 kDa (the chemokine SDF-1), and can detect with a sensitivitydown to 20 pM (with MAbs). Importantly, we are able to detect binding ofthe HIV-1 Envelope protein gp120 to one of its receptors (CXCR4) and weare able to detect binding of whole virus (lipoparticles containingreceptors) to the biosensor chip surface conjugated with a MAb. One ofthe highlights of this optimization has been the enablement of the firstkinetic analysis of MAbs against membrane proteins (FIG. 3B), a majorapplication of the BIACORE biosensor that was previously limited tonon-membrane protein targets. In this example, nine different monoclonalantibodies against GPCRs were characterized for their on-rate, off-rate,and affinity.

Example 32 Creation of Sensory Biosensor

This Example involves the creation of a lipoparticle biosensor, suchthat known membrane proteins having functions known or suspected to beinvolved in the senses of smell and taste are incorporated into thearray. The receptors that mediate the senses of taste and smell fallinto the category of membrane proteins, primarily G-protein coupledreceptors and ion channels. Such a tool will have extensive applicationsfor diagnostics, biodefense, food quality, water safety, and narcoticsdetection. A lipoparticle biosensor is constructed that contains allknown membrane proteins involved in the senses of taste and smell, witheach receptor being incorporated into a lipoparticle and thelipoparticle biosensor being composed of many such lipoparticles. Asrelated membrane proteins are discovered, they will also be incorporatedinto the lipoparticle biosensor.

The creation and screening of the Sensory Biosensor will closelyparallel the methods described in Example 11. First, lipoparticles areproduced containing known membrane proteins having functions known to beinvolved with taste and smell. Second, these lipoparticles are attachedto a biosensor surface. Finally, this lipoparticle biosensor will bescreened using samples to determine the presence of constituentmolecules that stimulate specific taste and smell receptors. The resultof this Example will be a tool to aid in the rapid testing of multiplesamples for a large number of potential stimulants. One skilled in theart would also recognize that a similar system could also be used fordetection of contaminants or components of other liquids, gases,beverages, foods, chemicals, perfumes, cosmetics, alcoholic beverages,narcotics, or aqueous solutions.

Example 33 Effect of Additives on Antibody Binding to Lipoparticles

The effects of additives on the binding of antibodies to a membraneprotein in a lipoparticle were measured. 10,000 RUs of the CXCR4monoclonal antibody 12G5 were amine-coupled to the surface of a CM5 chip(available from BIACORE, Piscataway, N.J.) (Flow Cell 2, Fc2). Fc1contained 10,000 RUs of mouse IgG. The data were reference subtractedusing the signal from the mouse IgG surface. Additives were mixed withlipoparticles just before their injection across the biosensor surface.The large spikes were due to changes in refractive index between runningbuffer and sample solution magnified by the increased viscosity ofadditives. BSA was not used in these experiments. The additives haddifferent effects on the binding of the antibody to lipoparticlescontaining CXCR4 on their surface (data not shown).

Example 34 Detection Ability of Antibody Binding to Lipoparticles

To determine the ability of the biosensor to detect antibody binding tothe lipoparticles, different concentrations of antibody were screenedagainst a biosensor surface comprising attached lipoparticles containingCXCR4. Lipoparticles containing CXCR4 were coupled to the surface of aBIACORE biosensor C1 chip. Monoclonal antibody concentrations down to 20picomolar were detected (FIG. 3A). A plot of ‘apparent’ k_(on) v k_(off)for nine different (bivalent) monoclonal antibodies against CXCR4 andCCR5 was created (FIG. 3B). Each kinetic data point is derived from adilution series of the monoclonal antibody binding to attachedlipoparticles. Points falling on the same diagonal line have the sameK_(D). Each binding series fit a bivalent model, and K_(D) wascalculated using the ratio of k_(off) to k_(on).

Example 35 Incorporation of an Ion Channel

Membrane potential is generated and maintained by concentrationgradients of charged ions, as regulated by selective ion channels andtransporters. Potassium ion (K+) channels have been particularly wellstudied due to their primary importance in excitable cells (see (Deutsch(2002), Annu Rev Physiol, 64:19-46, Ford, et al. (2002), Prog Drug Res,58:133-168)). A number of toxins are known to bind specifically toK-channels, a bacterial K-channel has been crystallized, and a varietyof K-channels with various regulatory features have been defined andcharacterized. Drosophila Shaker (GenBank Accession Number M17211, GI:157063) is a voltage-regulated (opens upon depolarization) K-channelthat serves as a prototype ion channel herein due to its extensivecharacterization. Lipoparticles containing Shaker, as well as two otherion channels (Kv1.3 and CFTR) (FIG. 8) were produced as described hereinby co-transfecting 293 cells with plasmids encoding MLV Gag and the ionchannel under the control of CMV promoters.

Example 36 Detection of Membrane Potential

Like many ion channels, Shaker is a complex membrane protein, containingsix transmembrane domains and forming tetramers in the membrane (Deutsch(2002), Annu Rev Physiol, 64:19-46). The structural integrity of itsactive and non-active conformations is confirmed by ligand binding andfunctional assays. Even if the ion channel does conduct ions, it ispossible that it may conduct for too brief a period to be detected (e.g.through inactivation or ion depletion). To allow for this possibility,we will be using an inactivation-gate removed variant of Shaker. Variousmethods for detection are described herein to detect even brief changesin membrane potential (e.g. substrate injection, fast response probes).

The purity and concentration of membrane protein can be important indetermining sensitivity and signal-to-noise. For ion channels, in whicha hundred channels can be responsible for an entire cell signal,membrane protein density may not be a critical parameter. To test this,lipoparticles are prepared with high, medium, and low amounts of Shaker(on a relative basis) by varying the amount of DNA ranging from 5-40 μgper 10 cm dish used to prepare the lipoparticles. These preparations arethen tested to determine signal-to-noise and sensitivity to correlatefunctional response with protein density.

The lipoparticles were then loaded with the dye. Unlike water-solubledyes, membrane potential probes are lipophilic, simplifying theincorporation and use of the dye with lipoparticles. As previously used(Montana, et al. (1989), Biochemistry, 28:4536-4539, Rohr, et al.(1994), Biophysical Journal, 67:1301-1315, Venema, et al. (1993),Biochim Biophys Acta, 1146:87-96), the membrane potential probedi-4-ANEPPS was obtained as a powder, resuspended in ethanol, and addedto an aqueous solution containing lipoparticles to a final concentrationof 5 μM. The dye partitions into membranes nearly instantaneously, asshown in FIG. 9.

One skilled in the art would also recognize that additional changes inthe composition of the lipoparticle could be introduced in order tofacilitate ion channel measurement. Such changes can include changingthe internal ion concentration, changing the membrane lipids, modulatingfluorescent dye content, or modulating water content. For example,lipoparticles can be soaked in high potassium buffer for several days inorder to equilibrate the internal content of the lipoparticles with theion concentration of the outside buffer. Similarly, purified lipids canbe added to suspensions of lipoparticles where the lipids will partitioninto the lipoparticle membrane. One skilled in the art would alsorecognize that membrane enveloped viruses, such as influenza, could alsobe loaded with a membrane potential dye as described herein.

Example 37 Testing for Ion Channel Function

Experimental use of labeled lipoparticles containing functionalK-channels follows well-established protocols (Montana, et al. (1989),Biochemistry, 28:4536-4539, Rohr, et al. (1994), Biophysical Journal,67:1301-1315, Venema, et al. (1993), Biochim Biophys Acta, 1146:87-96).Shaker lipoparticles are purified and resuspended in low K-buffer (1 mMKCl in 10 mM Hepes, plus sucrose to maintain osmolarity). Lipoparticlesare aliquoted into 96-well plates just before use (100 μg/ml finalconcentration in 100 μl), and inhibitory toxins are incubated with theion channels for 30 min. Assuming that the lipoparticle has an internalK+ concentration less than 150 mM and that its lipid membrane is intactand impermeable to K+, addition of high K-buffer (150 mM KCl in 10 mMHepes buffer) will initiate depolarization. If no ion channel ispresent, membrane potential will be maintained as long as lipoparticlesremain intact (half life of several hours at 37° C.), and thus nofurther fluorescence change will be observed. If a functionalvoltage-regulated K-channel is present, the ion channel will open inresponse to depolarization and a change in fluorescence of di-4-ANEPPSwill occur.

96-well format fluorometry detectors are used. The most useful detectorspermit ratiometric detection of two emitted or excited wavelengths. Anexample detector is Wallac Victor® fluorescence detector. Due to the lowinternal volume of lipoparticles, transmembrane electrochemicalgradients are expected to reach equilibrium rapidly and with very smallion concentration changes. In order to capture even fleeting signalsgenerated milliseconds after addition, an automated injector is used toadd high-K buffer to samples. Lipoparticles containing high and lowquantities of ion channels for increased stability of signal are alsotested.

Failure to detect a specific signal can be categorized in two ways: nosignal or non-specific signals. In the first case, lack of a signal ismeasured as the same fluorescence before and after addition of high-Kbuffer, as compared to control lipoparticles lacking the ion channel ofinterest. Primary causes may be lack of correct protein conformation,rapid equilibration of potassium across the membrane, or low sensitivityof detection. In the second case, a non-specific signal is measured asan increase in fluorescence in both experimental and controllipoparticles. Primary causes may be contaminating ion channels in thelipoparticles or leakiness of the lipoparticle membrane. These issuesare addressed with the use of contaminating protein inhibiting toxinsand impermeable ion substitutes, production from other cell types,control of buffer ionic strength, and, if necessary, direct measurementof the leakiness of the viral membrane. Because the absolute value (inmV) of membrane potential can be measured (see calibration withvalinomycin, below), no membrane potential fluorescence (0 mV) can bedistinguished from non-specific high membrane potential fluorescence.

The level of fluorescence detected from membrane potential dyes orfluorescent probes is calibrated to measure the absolute value ofelectrical membrane potential in mV. Absolute measurements are importantas an internal control, for comparison of inhibitors, toxins, and newion channels, and for comparison to others' results using differentdetection systems. The most widely used calibration procedure is basedon membrane potential clamping to a potassium equilibrium diffusionpotential. Saturating amounts (1 μM) of valinomycin is added to cells atgradients of potassium solution from 0-150 mM. A calibration curve isplotted to correlate fluorescence to membrane potential, as calculatedfrom the Nernst equation. One skilled in the art would also recognizethat membrane enveloped viruses, such as influenza, could also be testedfor ion channel function as described herein.

Example 38 Ligand Binding

A prerequisite for ion channel function within lipoparticles is thecorrect conformation of the ion channel. Table 12 lists some toxins andthe ion channels to which they bind. Toxins that bind to Shaker havebeen well characterized for their ability to bind under definedconditions and in defined conformational states; in many cases, bindingcan distinguish active vs. non-active conformations.

TABLE 12 Inhibitors of K-channels Toxin Binding/Blocking ConditionsCharybdotoxin Blocks Kv1.3 and Shaker Binds strongly (10 pM) to closedK-channels (−70 mV) Ca inhibits binding Low ionic strength, high pH(8.5) enhances binding Alpha-Dendrotoxin Blocks Kv1.3 and Shaker Bindsstrongly under low ionic strength conditions Binds very weakly underhigh ionic strength conditions Agitoxin-2 Blocks Kv1.3 and ShakerMargatoxin Blocks Kv1.3, not Shaker Iberiotoxin Blocks Ca-activatedK-channels, not Kv1.3 or Shaker

Charybdotoxin (CHTX), a 37 amino acid (4.3 kDa) peptide purified fromscorpion venom can be used as one method to demonstrate that the Shakerchannel is conformationally intact within lipoparticles. CHTX is ahighly specific, potent, and impermeant blocker of both Shaker andKv1.3, and is resistant to functional degradation under in vitro cultureconditions. Ionic conditions strongly influence the ability of CHTX tobind and block (see table above) and can serve as additional specificitycontrols. Other controls can include, for example, identicallipoparticles that contain an unrelated receptor or no receptor. CHTXand additional toxins of known specificity (dendrotoxin, iberiotoxin,margatoxin, and others as necessary) can be used within the followingbinding experiments:

Biosensor Binding. Lipoparticles containing Shaker are covalently boundto BIACORE biosensor chips and used to detect direct binding of ligandsto the membrane protein.

Direct Binding. Labeled toxins (radioactive or fluorescent) are used toassess direct interaction with the Shaker ion channel. Direct binding oflabeled toxins can be readily performed using a filtration assay withminor modifications, similar to experiments we have performed previously(Baik, et al. (1999), Virology, 259:267-273, Deutsch, et al. (1991), J.Biol. Chem., 266:3668-3674, Doranz, et al. (1999), J. Virol.,73:10346-10358, Doranz, et al. (1999), J. Virol., 73:2752-2761). Incases where labeled toxins are not available, unlabeled toxins are usedto competitively inhibit binding of a labeled toxin.

Example 39 Detection within Microfluidic Devices

The ability to present structurally intact, functional ion channelswithin a 100 nm particle allows more advanced technologies to be usedfor drug discovery against them. Lipoparticles are used within severalmicrofluidic devices to test their compatibility for the detection offunctional response. Multiplexed microfluidic devices with thecapability of detecting hundreds to thousands of samples simultaneouslywill be preferred.

Detection of ion channel activity is performed on a Caliper 250workstation. 2×10⁷ lipoparticles are loaded into wells of a LabChip™.The lipoparticles are labeled by loading membrane potential indicatorsinto a well of the LabChip and the Caliper 250 mediates mixing andintegration of dyes into the lipoparticle membrane. Various bufferscontaining PBS alone, and PBS containing agonists/antagonists asdescribed herein are utilized to determine specificity and activity ofthe ion channels. The agonists/antagonists are titered to determine adose response curve for each molecule. The Caliper 250 performsscreening of the samples by mixing small volumes of lipoparticles (>50ul) with the various buffer solutions and detecting changes influorescence associated with a change in membrane potential eluded to bychanges in fluorescence of indicators of membrane potential integratedinto the lipoparticles as described above. Ligands and toxins used forexperimentation include but are not limited to those described above. Inaddition, antagonists and toxins with known activity such as thoseidentified herein can be used as a control for ion channel blocking.Thereafter, high throughput screening applications are established toobserve the effects of various compounds on ion channel activation andthus membrane potential changes. One skilled in the art would recognizethat additional detection devices, including microfluidic devices, flowcytometry, a 96-well plate, a 384-well plate, a 1536-well plate, a glassslide, a plastic slide, an optical fiber, a flow cytometer, amicroscope, a fluorometer, a spectrometer, or a CCD camera could also beused.

Example 40 Subcellular Detection

Lipoparticles are used as nanometer-sized sensors of ions and voltage toprobe the synaptic junctions and intracellular compartments of, forexample, living neurons. Fluorescent measurements are taken using amicroscope in both resting cells (absolute levels of ions or membranepotential) and in cells responding to stimuli (relative changes). As amodel system, primary neurons or N-tera 2 (NT2, Stratagene CloningSystems) cells are used, which can be differentiated into neuron likecells that contain all of the physical properties of neurons and can begrown in culture. Lipoparticles are labeled with membrane potential dyeas described in Example 35. The lipoparticles are microinjected eitherinto or onto the NT2 cells or neurons. The cells or neurons areactivated using electric charge to artificially depolarize the membranethus causing activation. Alternatively, the NT2 differentiated neuronsare activated by addition of 100 μM γ-aminobutyric acid (GABA) orN-methyl D-aspartate (NMDA). Both of these neurotransmitters have beenshown to cause membrane depolarization in neurons using patch clamptechniques. Activation of the NT2 cells causes changes in membranepotential and ion concentration in areas around the cell that isdetected using lipoparticles containing reactive fluorescent dye.Organelles such as mitochondria can also be monitored by targetinglipoparticles to the mitochondria. In addition, membrane potential isobserved near the site of neurotransmitter release, using fluorescentlipoparticles responsive to membrane potential to determine how membranepotential affects vesicle fusion events.

Example 41 Cell Based Detection

Nanometer-sized sensors of ions and voltage are used to probe membranepotential of various cells upon activation. Lipoparticles containingmembrane potential dyes are attached to membranes of Jurkat T-cells(ATCC) and primary T-cells isolated from normal human volunteers. TheT-cells are activated using antibodies targeted against CD3, a member ofthe T cell receptor complex that induces cell activation. In addition,SDF-1alpha, a ligand for CXCR4, is also used to observe cellularactivation states upon stimulation of CXCR4, thus mimicking some aspectsof HIV infection. Observation is performed using fluorescence microscopyto verify expected changes in lipoparticle fluorescence. Subsequently,microfluidic devices such as those described herein will be utilized forHTS of inhibitors of activation.

Example 42 Amino Acid Transporters and Microfluidic Devices

Lipoparticles are used to monitor the ability of amino acid transportersto move amino acids across a membrane. Specifically, MCAT-1 isincorporated into a lipoparticle(s). Microfluidic devices describedherein are employed to detect transporter activity. MCAT-1 containinglipoparticles are loaded into a well of a LabChip. In another well,tetramethylrhodamine (TR) is loaded and subsequently mixed with thelipoparticles whereby the TR is loaded into the lipoparticle.Fluorescein (FL) tagged amino acids are added into wells of the LabChipfor eventual mixing with lipoparticles. In addition, MCAT-1 agonists andantagonists are loaded into wells of the LabChip. The Caliper 250mediates mixing of the lipoparticles with the TR. Once the TR hasentered the lipoparticle, they are mixed with either control buffer orbuffer containing an agonist or antagonist of MCAT-1. Once this processis finished, the lipoparticles are exposed to the FL tagged amino acids.The ability of MCAT-1 to mediate transport of the amino acids into thelipoparticle is evaluated by measuring the resonance energy transfer(RET) between the FL-amino acids and the TR in the lipoparticle. RET isa very sensitive technique to measure interaction between participatingfluorophores. Therefore, minute changes in RET determines the efficiencyof transport across the lipoparticle membrane. Different pairs offluorophores capable of participating in RET can also be used todetermine presence of amino acids that have been transported.

Example 43 Amino Acid Transporters

Lipoparticles are used to monitor the ability of amino acid transportersto move amino acids across a membrane. MCAT-1 is incorporated intolipoparticles. MCAT-1 containing lipoparticles are placed in aneppendorff tube. Radioactive amino acids are added into tubes with thelipoparticles. A buffer containing cofactors necessary for MCAT-1activity (e.g. ATP) is also added. The lipoparticles are incubated withthe radioactive amino acids for 1 hour and then separated from freeamino acids by spinning the lipoparticles through a sucrose cushion. Theability of MCAT-1 to mediate transport of the amino acids into thelipoparticle is evaluated by measuring the radioactivity remaining inthe lipoparticles. Lipoparticles without MCAT-1 will serve as negativecontrols. On skilled in the art would also recognize that other types ofdetection could also be used, such as the incorporation of fluorescentamino acids.

Example 44 Fluorescent Dye Incorporation into Lipoparticles

Lipoparticles containing the fusion protein CXCR4-GFP were generated bymethods described herein. The lipoparticles were imaged by fluorescencemicroscopy to visualize the expression of the protein and to visualizethe lipoparticles. FIG. 9A demonstrates the signal generated byCXCR4-GFP. To determine if a fluorescent dye is capable of beingincorporated into a lipoparticle DI-4 ANEPPS was added to thelipoparticles. As discussed above, the dye does not fluoresce unless itis incorporated into lipids. FIG. 9B demonstrates that the lipoparticlescan be visualized after adding DI-4 ANEPPS, thereby indicating that thedye has been incorporated into the lipoparticle. For comparison, 0.2micron YG Fluoresbrite beads (Polysciences, Inc.) were imaged at thesame magnification as the other two panels (FIG. 9C).

Example 45 Using a Lipoparticle to Generate an Immune Response in aMouse

Lipoparticles were used to generate polyclonal antibodies against theGPCR chemokine membrane proteins CCR5 and CXCR4. CCR5 and CXCR4 arechemokine receptors that are also used by HIV as a cellular receptor(Berger, et al. (1999), Annu. Rev. Immunol., 17:657-700). The chemokinereceptors CCR5 and CXCR4 have been well studied and have been shown tobe involved in HIV infection, hematopoiesis, breast cancer metastasis,stem cell migration, neuronal development, and rheumatoid arthritis. Inorder to better understand their structure and function, both of thesereceptors have previously been incorporated into lipoparticles (Endres,et al. (1997), Science, 278:1462-1464, Hoffman, et al. (2000), Proc.Natl. Acad. Sci. USA, 97:11215-11220).

The production of lipoparticles containing CCR5 and CXCR4 has beenpreviously described (Hoffman, et al. (2000), Proc. Natl. Acad. Sci.USA, 97:11215-11220). Briefly, 293T cells were transfected overnightwith 120 μg of plasmid expressing the receptor CCR5 or CXCR4 and 30 μgof pCGP (encoding for MLV-gag/pol under the control of theCMV-promoter). The following morning, the medium was replaced with freshmedia supplemented with 10 mM sodium butyrate (an up-regulator of theCMV-promoter). Supernatants were harvested after approximately 2 days.Lipoparticles were purified by filtration of supernatants through a 0.45μm filter and ultracentrifugation through a 20% sucrose cushion. Todetect incorporated proteins, particles were analyzed by Western blotusing an anti-MLV-gag antibody to detect the primary structural proteinof the virus and anti-receptor antibodies to detect the receptor ofinterest or a tag on the receptor. For the purposes of the presentexperiments, lipoparticles were produced using human HEK-293 cells.HEK-293 cells are a human embryonic kidney cell line. Lipoparticles canalso be produced in murine 3T3 cells or retroviral packaging cells (e.g.PA317 cells) that constitutively produce MLV virus in order to producean antigen using cells syngeneic to the immunized host.

Two mice were immunized for each receptor (4 mice total). Each mouse wasimmunized with approximately 100-200 μl of sample at a concentration of0.5 mg/ml by injecting mice intraperitoneally and then boosting threetimes at days 14, 21, and 49 each with 50-100 μl additionallipoparticles at the same concentration. The dosage was based on totalviral particle protein. No adjuvant was used in initial experiments inorder to retain the complete structure of the lipoparticles and membraneproteins.

The sera from each mouse was collected by retro-orbital bleed andscreened by flow cytometry and western blot. Sera fromlipoparticle-immunized mice were first analyzed by flow cytometry toidentify their reactivity against parental HEK-293 cells. Flow cytometryresults demonstrated that a strong immune response was generated againstthe surface molecules of the parental HEK-293 cell. As little as a1:100,000 dilution of mouse serum was sufficient to stain the surface of293 cells (FIG. 10A). Testing of cell types not involved in theimmunization indicated little or no cross-reactivity (FIG. 10B),indicating that the response was specific to molecules from the 293cell-derived lipoparticle surface. Flow cytometry analysis of sera fromlipoparticle-inoculated mice also revealed that antibodies weregenerated against the membrane protein of interest (FIGS. 11 and 12).

Western blot is an alternative method of analyzing a sera response.However, detection is limited to linear (non-conformational) epitopesbecause the proteins are denatured during analysis. Western blotanalysis of sera from lipoparticle-immunized mice showed littlereactivity with 293 cell lysate or other (QT6 cell) lysates (FIG. 13).ELISA results against Triton-disrupted 293, NIH-3T3, CHO, and QT6 celllysates were similarly non-reactive with mice sera (data not shown). Thelack of response to 293 cell lysates suggests that antibodies againstundesired intracellular proteins from producer cells were not generated.However, western blot (FIG. 13) and ELISA (FIG. 14) did reveal a largeresponse against the Gag structural protein of the lipoparticle. Theantibody response to Gag does not interfere with hybridoma screeningbecause cells do not naturally express Gag.

In some of the lipoparticle inoculations, CXCR4 with a C-terminal HA tag(CXCR4-HA) was used. The resulting sera demonstrated a clear reactivitywith the HA-tagged protein (FIG. 4) but not with the untagged protein,indicating that sufficient CXCR4-HA protein was present in thelipoparticle preparations to generate antibodies against it.

Polyclonal antibodies directed against CCR5 and CXCR4 are thusgenerated. Standard CCR5 and CXCR4 polyclonal antibodies are used forinhibition of HIV infectivity, blocking of chemokine binding andsignaling, localization of receptors during intracellular trafficking,detection of expression in cells and tissues, and blocking of HIV gp120binding.

Example 46 Generation of Monoclonal Antibodies Using an AntigenicComposition Comprising a Lipoparticle

The production of lipoparticles is discussed herein and the immunizationprotocol for the generation of polyclonal antibodies as described aboveis followed.

The sera from each mouse is screened for antibody production. Flowcytometry can be used to determine reactive sera. Stably transfectedcell lines expressing CCR5 or CXCR4 are used for detection.Alternatively, sera can be screened against cells expressing either CCR5or CXCR4 in a cell-ELISA format (CELISA) as described herein. Thespleens from the mice giving the strongest response are harvested, andthe B cells therefrom are fused with murine myeloma cells to formhybridomas following standard protocols (Harlow, E. and D. Lane.Antibodies: A laboratory manual. Cold Spring Harbour Laboratory Press.1988).

The resulting hybridomas are screened and those exhibiting antibodyproduction are diluted to achieve single-cell isolation and grown toproduce enough supernatant for screening antibodies. Hybridomasupernatants are screened using CELISA as the first test to detectnative cellular expression of the receptor on human cells. Briefly,stably transfected cells expressing either CCR5 or CXCR4 are adhered toan ELISA plate and incubated with supernatant from each hybridoma at a1:5 dilution. Binding is detected with GAM antibody. To verifyreactivity, Mabs are screened using untransfected cells as a negativecontrol.

In some embodiments, where stable cells are not available for thecellular protein of interest, a number of alternate screening mechanismsare employed on an as-needed basis. First, transiently transfected cellsare employed because cells can be prepared with minimal effort and forwhich prolonged expression is not necessary. This can be particularlyuseful if the protein of interest is toxic. Second, inducible cell linescan be prepared that express the receptor of interest under an induciblepromoter. Third, the hybridomas are screened against lipoparticlesthemselves. In particular, sera from mice immunized with lipoparticlesare screened against lipoparticles containing the same protein. Finally,cells can be permeabilized to allow access to intracellular epitopes.

The hybridomas having reactive supernatants are then expanded in numberand are typed for immunoglobulin class. The antibodies can be producedin milligram quantities by injecting the hybridomas in miceintraperitoneally and harvesting the ascites fluid.

Similar to as was performed above, Western blotting is used to determineif Mabs are directed to linear epitopes of receptors. Lysates of cellsexpressing CCR5 or CXCR4 are run on SDS-PAGE under denaturingconditions, transferred to PVDF membranes, cut into strips, and reactedwith Mabs. Antibodies to linear epitopes of CCR5, Mabs to epitope tagson CCR5 and CXCR4, and mouse and rabbit seras previously made againstCCR5 and CXCR4 serve as positive controls. Reactivity of a Mab to areceptor by Western blot is indicative of recognition of anon-conformational, linear epitope within the receptor. Previous Mabsdirected to linear epitopes against CCR5 (e.g. Mabs CTC5 and CTC8) areall directed to the first thirteen amino acids of the receptor (distalN-terminus), and were all produced by immunization with cells expressingCCR5 in the presence of Freund's adjuvant (Lee, et al. (1999), J. Biol.Chem., 274:9617-9626). Previously, only one Mab to a linear epitope ofCXCR4 (4G10) has been isolated, and that Mab was produced using anN-terminal peptide.

Flow cytometry is used to complement CELISA in determining which Mabsare best for detecting antigen on the cell surface. Stably transfectedcell lines expressing CCR5 or CXCR4 are used for detection. Differentconformations of CCR5 and CXCR4 can be expressed on different celllines, and many Mabs, such as 12G5 against CXCR4, are capable ofrecognizing only a subset of these conformations (Baribaud, et al.(2001), J. Virol., 75:8957-8967, McKnight, et al. (1997), J. Virol.,71:1692-1696). Therefore, cell lines that express CCR5 or CXCR4endogenously (e.g. PM1 cells and HeLa cells, respectively) are testedagainst select Mabs for reactivity within different cellular contexts.

The structure and function of over one hundred CCR5 and CXCR4 mutationshas previously been characterized, including CCR5 N-terminaltruncations, CCR5-CCR2 receptor chimeras, CCR5 mutations thatindividually change each extracellular charged amino acid to Alanine,CXCR4-CXCR2 chimeras, and CXCR4 point mutations (Blanpain, et al.(1999), J. Biol. Chem., 274:34719-34727, Doranz, et al. (1997), J.Virol., 71:6305-6314, Doranz, et al. (1999), J. Virol., 73:2752-2761,Edinger, et al. (1997), Proc. Natl. Acad. Sci. USA, 94:4005-4010, Lu, etal. (1997), Proc. Natl. Acad. Sci. USA, 94:6426-6431, Rucker, et al.(1996), Cell, 87:437-446). These genetic panels are used to characterizethe antigenic structure of the Mabs that are isolated using lipoparticleimmunizations. In particular, receptor chimeras are used to quickly andaccurately identify CCR5 and CXCR4 domains that are involved in Mabbinding.

CELISA using transiently transfected cells facilitates the rapidscreening of large numbers of Mabs against a large number of mutationsor conformations of a protein in a 96-well format. Flow cytometry can beused to test some Mabs for reactivity against select mutations orconformations of a protein, for example, to identify the epitope thatthe antibody recognizes. Quantitative flow cytometry, which candetermine the affinity of a Mab for a receptor using quantitativestandards, can also be employed to distinguish antigenic reactivity fromaffinity.

Example 47 Epitope Mapping

Epitopes of several dozen Mabs against CCR5 and CXCR4 that have beenproduced using various immunization protocols have been previouslymapped (Baribaud, et al. (2001), J. Virol., 75:8957-8967, Lee, et al.(1999), J. Biol. Chem., 274:9617-9626). Each of these antibodies can becompared to antibodies produced using lipoparticles. These antibodieswere mapped using the same panels of CCR5 and CXCR4 mutants identifiedabove, so competition with lipoparticle-derived Mabs provides aconfirmation of epitope reactivity.

Competition assays are used to determine the Mabs that have unique oroverlapping epitopes with the Mabs currently available. These assays aredone by CELISA in 96-well format for high throughput mapping usingbiotinylated Mabs. Cells expressing either CCR5 or CXCR4 are incubatedwith a Mab produced according to the present invention, and then abiotinylated Mab that has been previously characterized is used to bindto the same cells. The inability of a biotinylated Mab to bind wouldsuggest that the test Mab was bound to the same or an overlappingepitope used by the biotinylated Mab.

Panels of CCR5 and CXCR4 Mabs were previously tested in functionalassays to determine their ability to block ligand binding and virusinfection (Baribaud, et al. (2001), J. Virol., 75:8957-8967, Lee, et al.(1999), J. Biol. Chem., 274:9617-9626). Specifically, the ability ofthese panels to block binding of radiolabeled chemokines (¹²⁵I-labeledRANTES and MIP-1α against CCR5, and SDF against CXCR4), binding ofradiolabeled HIV gp120 (¹²⁵I-labeled JRFL and CM235 against CCR5, andHXB against CXCR4), signaling of chemokines (chemotaxis assays using SDFand CXCR4-containing cells) and entry of HIV virus (JRFL, ADA, and BaLagainst CCR5, HXB against CXCR4) was assessed. Standard CCR5 and CXCR4Mabs are utilized for mapping of receptor structures, inhibition of HIVinfectivity, blocking of chemokine binding and signaling, localizationof receptors during intracellular trafficking, detection of expressionin cells and tissues, and blocking of HIV gp120 binding. Mabs generatedaccording to the present invention allow similar questions to beaddressed for different membrane proteins.

Example 48 Making Mabs Against a Multimeric Membrane Protein

For Mab production against a complex single-TM protein, thesingle-spanning membrane protein DC-SIGN is used. DC-SIGN has beencharacterized extensively. DC-SIGN is a type II integral membraneprotein that is expressed at high levels on dendritic cells (DCs) andthat functions as a homotetramer. A related receptor, DC-SIGNR, isexpressed on endothelial cells in the liver, lymph node sinuses,placenta, and intestine. DC-SIGN binds to both ICAM-2 and ICAM-3, and inso doing mediates interactions between dendritic cells and endothelialcells and T-cells, respectively. In addition, HIV and SIV bind with highaffinity to DC-SIGN, and this binding event appears to account in largemeasure for the ability of DCs to efficiently mediate infection ofT-cells. It has been proposed that HIV-DC-SIGN interactions may alsoplay a role in sexual transmission of virus. After crossing theepithelium, HIV encounters immature, DC-SIGN positive DCs in thesubmucosa. If HIV binds to these cells, it is believed to be transportedto regional lymph nodes as a consequence of normal DC trafficking, thusdelivering infectious virus to the major site of HIV replication invivo. To test this model, antibodies to DC-SIGN are generated thatpotently inhibit virus binding to DC-SIGN. Such antibodies can be usedin the rhesus macaque model to determine if they impact sexualtransmission of either SIV or SHIV in this animal model (SHIVs are SIVparticles that bear an HIV envelope protein).

Mabs to DC-SIGN are generated as described herein. Briefly,lipoparticles containing DC-SIGN are produced and used to immunize mice.The sera is screened for reactivity and hybridomas established forantibody production.

Example 49 Making Mabs Against an Ion Channel Protein

Kv1.3 is a well-studied type of potassium channel (K-channel) thatactively transports potassium ions across the cell membrane. Kv1.3 isexpressed in T-cells, and inactivation of this channel results in thesuppression of T-cell activation. A specific inhibitor of Kv1.3 would beuseful as a therapeutic immunosuppressant. Kv1.3 spans the lipid bilayersix times and functions as a tetramer in the membrane. Kv1.3 is highlyconserved and the majority of the protein is buried in the lipid bilayer(Doyle, et al. (1998), Science, 280:69-77), explaining why only ahandful of Mabs have ever been elicited to any K-channel and those fewMabs have been generated using peptides and fusion proteins.

Mabs to Kv1.3 are generated as described herein. Lipoparticlescontaining Kv1.3 are produced and used to immunize mice. The sera isscreened for reactivity and hybridomas established for antibodyproduction.

The ability of Kv1.3 to form homotetramers has been well-studied, andmutations in T1, its tetramer-formation domain, eliminate its ability totetramerize (Doyle, et al. (1998), Science, 280:69-77, Lu, et al.(2001), Biochemistry, 40:10934-46). By producing cells which expresseither wild-type Kv1.3 or the mutant non-oligomerizing form of Kv1.3,the Mabs with reactivity against the monomer and the tetramer can beproduced. Mabs that have proven reactive against tetramers of theK-channel (the primary screen) are screened against cells that expressmonomeric Kv1.3. Mabs that react with tetrameric but not monomeric Kv1.3are assumed to recognize epitopes in Kv1.3 that are formed when theprotein oligomerizes. Such Mabs would be highly valuable in determining,among other questions, when during the synthesis of Kv1.3 within thecell does oligomerization and functional activation occur.

Example 50 Making Mabs Against ErbB2 and ErbB4

Members of the Epidermal Growth Factor (EGF) family of receptors aresingle transmembrane Tyr kinase proteins that operate as homo-dimers orheterodimers. Members of this family have been implicated in tumorgrowth, and the combination of receptors expressed within a tumor hasbeen shown to have an effect on the severity of the tumor. For example,in medulloblastomas, the expression of ErbB2 and ErbB4 has a worseprognosis for the child than if the tumor only expressed one of thesereceptors. This raises the possibility that the heterodimer of thereceptors is forming in the more deadly tumors (see, for example,Gilbertson R J, Perry R H, Kelly P J, Pearson A D, Lunec J. (1997),“Prognostic significance of HER2 and HER4 co-expression in childhoodmedulloblastoma.” Cancer Research August 1; 57(15):3272-80).

By expressing different combinations of Erb receptors in lipoparticles,the generation of Mabs specific to a certain combination of subunits ispossible. Screening the Mabs is accomplished using CELISA with cellsthat express either a single Erb receptor subunit forming homodimericreceptors or with cells expressing known combinations of receptorsubunits which will form heterodimers. A Mab for a specific combinationwill bind to cells expressing that combination but not to cellsexpressing only one type of subunit or a different combination ofreceptor subunits.

Mabs that can distinguish between the heterodimer and the homodimers ofthese receptors are useful to clinically test the combinations of thesereceptors in tumors, and possibly help direct specific therapies.

Example 51 Making Mabs Against a Constitutively Active Receptor

Monoclonal antibodies are generated against mutant constitutively activereceptors following a procedure that is similar to examples herein.Cysteine substitutions in the extracellular regions of the ErbB4receptor result in a constitutively active form of the receptor.Lipoparticles containing this mutant are used to generate Mabs againstthe active form of the receptor.

Example 52 Using Lipoparticles to Generate Antibodies Against Proteinswhere Conformation is Less Important

Lipoparticle are used as an antigen delivery tool for single membranespanning proteins or tethered soluble proteins.

Proteins that span the membrane once (CD4, Neuropilin, and Plexin-2) canalso be embedded within lipoparticles (Hoffman, et al. (2000), Proc.Natl. Acad. Sci. USA, 97:11215-11220). However, single-TM proteins areless dependent on a lipid membrane for their conformation; many cansimply be truncated just before their transmembrane domain in order tocreate a soluble form of the protein that often retains the sameextracellular structure as the intact protein.

Polyclonal and monoclonal antibodies to the single transmembrane proteinCD4 are generated as described in Examples 1 and 2. Briefly, cellsover-expressing CD4 are used to produce lipoparticles containing CD4.These particles are used to immunize mice. The sera is screened forreactivity and either polyclonal antibodies will be collected orhybridomas established for monoclonal antibody production. Theantibodies generated will be screened as described herein.

The CD4 antibodies generated are compared to other available antibodiesin a variety of tests including binding, cross-reactivity, and efficacyin inhibiting an immune response.

Example 53 Making an Antibody Against a Non-Membrane Protein

Another application for the lipoparticle is to link soluble proteins tothe membrane of the lipoparticle in order to generate a Mab. Resistin isa soluble protein derived from adipocytes. In mice, Resistin has beendemonstrated to cause insulin resistance and glucose intolerance,symptoms which are stereotypic of Non-Insulin-Dependant DiabetesMellitus (NIDDM, or Type 2 diabetes). The administration ofanti-resistin antibodies have been demonstrated to improve blood sugarand insulin action in obese mice. Generation of a variety of human Mabsto Resistin may help patients of Type 2 diabetes.

Resistin is tethered to the lipoparticle in several ways. First,Resistin is linked to phospholipids-poly(ethylene glycol) (PL-PEG) (Wonget al. Science, 1997, 275:820-822; L1 and Kao Biomacromolecules. 2003July-August; 4(4):1055-67). Incubation of Resistin-PL-PEG withlipoparticles results in coating of the lipoparticles with Resistinextending from the membrane.

Alternatively, a fusion protein of a transmembrane domain (thetransmembrane domain of CD4) and Resistin is expressed in a 293 cellline. Lipoparticles are produced from these cells which have Resistin onthe exterior of the membrane. Additionally, Resistin can be linked to aGPI anchor and expressed on cells for lipoparticle production.

The Resistin-linked lipoparticles are injected into mice as describedherein. Polyclonal and monoclonal antibodies to Resistin-linkedlipoparticles are generated as described in examples herein. Briefly,lipoparticles containing linked-Resistin are produced and used toimmunize mice. The sera is screened for reactivity and either polyclonalantibodies will be collected or hybridomas established for monoclonalantibody production.

Example 54 Using Lipoparticles to Make Specific Antibodies to ActivatedReceptors

CCR5 is incorporated into lipoparticles as described herein for othermembrane proteins. Lipoparticles containing CCR5 are incubated withMIP1α (ligand to CCR5) resulting in the binding of the ligand to thereceptor. Unique epitopes formed by the bound receptor are present.

As described herein, the lipoparticle with the bound receptor-ligandcomplex is used to generate an immune response in mice. This isaccomplished by using the lipoparticles directly after incubation andrelying on the interaction between the receptor and the ligand to bemaintained during the immunization.

Alternatively, after incubation with the ligand, thelipoparticle/receptor/ligand complex is cross-linked in order tocovalently bind the receptor to the ligand. Determination of thecross-linker is dependent on the specific functional groups and can bedetermined empirically. A water-soluble membrane impermeablecross-linker is used because the interaction between the ligand and thereceptor is outside the membrane. This technique results in a strongerbond between the receptor and ligand. Initially, 5-10 mM EGS (ethyleneglycol bis[succinimidylsuccinate]) is combined with the ligand and thereceptor lipoparticle for 30 minutes at 37° C. The cross-linkedlipoparticle/receptor/ligand complex is kept on ice until injected intothe animal for antibody production.

CELISA, as described herein, is used to screen seras, with theadditional step of screening the antibodies against cells expressing thebound receptor-ligand complex. Antibodies specific to bound receptorshould be positive in the presence of MIP1α but negative in cells whichhave the receptor but have not had ligand presented.

Alternatively, MIP1α is linked to PL-PEG as described in Wong et al(1997) Science 275, 820-823. MIP1α-PL-PEG is incubated withlipoparticles containing CCR5. During this incubation the PL-PEGmolecule incorporates into the lipid membrane of the lipoparticle. Withthe MIP1α tethered to the lipoparticle, the interaction between theligand and receptor is likely and diffusion of the ligand away from thereceptor is unlikely. Antibodies are generated against the lipoparticlesas described above. CELISA is performed with the additional step ofadding MIP1α to one group of cells to be screened against. Antibodiesspecific to bound receptor are positive in the presence of MIP1α butnegative in cells which have not had ligand presented. Antibodies canfurther be screened against “bathed” lipoparticles that have beenpre-incubated with ligand from the above example.

Example 55 Generation of Antibodies Against Lipoparticles ContainingMembrane Proteins Produced from Natural Cell Sources

Antibodies are generated against naturally occurring membrane proteins.Lipoparticles will be produced as described above and using an Ad-Gagvector to deliver the structural protein of MLV as described in U.S.Provisional No. 60/491,477, however, no membrane protein is exogenouslyintroduced—the membrane protein is expressed naturally from the cellschosen for production. This allows the capture of naturally expressedmembrane proteins from desired cell types.

Neural stem cells are cells which have the ability to self-renew, andcan divide to produce all the cell types of the nervous system. Onedifficulty in neural stem cell biology has been the identification ofthese cells. The protein Nestin has been proposed to be a marker ofthese cells, yet Nestin is also expressed in other cell types such asastrocytes. At best, a sub-population of Nestin positive cells can actas neural stem cells. Similar problems exist with other neural stem cellmarkers, such as Musashi.

In order to generate lipoparticles from neural stem cells, flowcytometry is used to obtain a population of Nestin positive cells fromprimary culture. Lipoparticles from the Nestin positive cell membranesare produced by infecting with a semliki forest virus vector thatexpresses MLV Gag. As the Gag drives budding from the cells,lipoparticles are formed containing the membrane proteins of the Nestinpositive cells.

It can also be useful to generate antibodies which recognizesubpopulations within this group. Mabs are produced which recognize theproteins of the Nestin lipoparticles. These Mabs are screened againstcultures of Nestin positive cells. Mabs which identify subpopulationswithin these cultures are identified. These antibodies can then be usedto further employ flow cytometry analysis and subdivide the population.

Example 56 Using Lipoparticles to Generate Mabs Against ReceptorVariants

Antibodies to HHV8 ORF74 are generated. ORF74 is incorporated intolipoparticles for immunization. Cells expressing the activated (wildtype) conformation of the receptor are used to screen the Mabs thatresult from this immunization. Positively reacting Mabs arecharacterized for their ability to recognize active vs. non-activeconformations of the receptors by screening Mabs for reactivity againstcells expressing non-active forms of the receptor (G-protein couplingcan be destroyed in nearly any GPCR by changing the conservedAsp-Arg-Tyr (DRY) motif in the second intracellular loop). Mabs thatrecognize the active form of the receptor but fail to recognize thenon-active form of the receptor are assumed to have extracellularepitopes that are induced to form upon intracellular G-protein coupling.

To differentiate conformational changes induced by the mutations fromconformational changes induced by G-protein coupling, wild type ORF74 ischemically uncoupled from G-proteins (inhibitors such as pertussis toxinand cholera toxin can uncouple many GPCRs, and analogs of GTP, such asGTP-gamma-S, can also lock GPCRs in a coupled or uncoupled state). Inaddition, Mabs are screened against additional independent mutations(e.g. DRY-box mutants (Doranz, et al. (1997), J. Virol., 71:6305-6314,Doranz, et al. (1999), J. Virol., 73:2752-2761),) that fail to signal.As an alternative screening procedure, Mabs to inactive conformations ofreceptors are produced by immunizing with lipoparticles expressingnon-active forms of GPCRs, screening for reactivity against the same,and then counter-screening for Mab reactivity with constitutivelyactivated forms of the receptor.

Example 57 Generation of Antibodies for Specific Epitopes

The Mabs produced in the examples described herein have been directedagainst entire receptor structures such as the extracellular region ofCXCR4. In some cases, however, antibodies that react with all epitopesof the receptor are not desired. For example, of many Mabs targeted toCXCR4, only one has ever been isolated that targets the amino terminusof the receptor. That Mab was raised against a peptide sequence of theamino terminus, is not conformationally sensitive, does not blockchemokine binding or signaling, does not block HIV infection, and hasonly weak affinity (Baribaud, et al. (2001), J. Virol., 75:8957-8967).The amino terminus of CXCR4 has been shown by several groups to play anintegral role in chemokine interaction and HIV fusion. In addition, noMabs to the third extracellular loop of CXCR4 have yet been identified.Such Mabs could be useful as western blotting reagents, for detectingstructural changes in the receptors, for blocking HIV and chemokineinteractions, and for screening compounds to these molecules that targetspecific domains of the receptor. Traditional methods of Mab production,however, may never elicit such Mabs.

Therefore, Mabs targeting epitopes of CXCR4 are generated usingmutations to target epitope reactivity. Specifically, CXCR4-CXCR2chimeras are used in which each domain is substituted individually or incombination with the equivalent domain of the other receptor (Baribaud,et al. (2001), J. Virol., 75:8957-8967, Doranz, et al. (1999), J.Virol., 73:2752-2761, Lu, et al. (1997), Proc. Natl. Acad. Sci. USA,94:6426-6431). A chimera that contains the amino-terminus of CXCR4 butthe extracellular loops of CXCR2 (a chimera termed 4222) has beenpreviously created (Doranz, et al. (1999), J. Virol., 73:2752-2761). Anequivalent panel with CCR5-CCR2 chimeras can also be used. To screen forCCR5 or CCR2 epitope-reactive antibodies.

Such chimeras are used to produce Mabs against specific domains ofdesired receptors. For example, lipoparticles are produced containingthe chimeric receptor 4222 and these lipoparticles are used forimmunization. The resulting Mabs are screened against wild type CXCR4,and positive clones will be counter-screened against the backgroundparental receptor, CXCR2. Mabs that are raised against 4222 andrecognize CXCR4 but do not recognize CXCR2 are presumed to be directedto the amino terminus of CXCR4. Mabs that react with both receptors arelikely to recognize conserved structures within this chemokine receptorfamily. Additional point mutations of CXCR4 can be used to map the exactresidues of reactivity within CXCR4.

Example 58 Improvement of Antibody Elicitation Using Different Methodsof Production and Immunization

To determine optimal conditions to generate antibodies and elicit immuneresponses, various conditions are modified. A wider range of doses forinitial immunization and subsequent boosts, the use of adjuvant, and theroute of immunization is explored. Results from these experiments enableone to base future immunizations on protocols that have been wellcharacterized, rather than the hit-or-miss approach to generating usefulMabs to complex receptors that is often taken. The experiments aresummarized in Table 13.

TABLE 13 Summary of varied conditions Group Prime Boost Route Adjuvant 1100 μg 3 × 50 ug IP Ribi 2 250 μg 3 × 100 ug  IP Ribi 3 100 μg 3 × 50 ugFootpad Ribi 4 100 μg 3 × 50 ug IP TiterMax 5 100 μg 3 × 50 ug IP None

Two mice are used for each group above, and the co-receptor CCR5 is usedas the prototype receptor in these experiments. In most cases hybridomasare not made; sera reactivity (titered dilutions) is adequate todetermine the quantitative increase in reactivity using differentimmunization protocols. In the case of adjuvant, a fundamentaldifference in the type of Mab produced (e.g. linear or non-linearepitope) is determined by competition of sera with CCR5 Mabs of knownreactivity, and by Western analysis of denatured protein as described inExample 45.

Intraperitoneal (IP) injection, intravenous (IV) immunization,subcutaneous (SC), and footpad injections (FP) have all been usedsuccessfully for the isolation of Mabs to complex receptors. In order tocompare these routes of immunization, mice are injected withlipoparticles IP and FP, the two routes that have worked best in thepast. Sera from these mice are compared for quantitative reactivity bytitering the seras and comparing to a standard for relative reactivity.

The number of lipoparticles can also be tested. Based on calculationscomparing lipoparticles to cellular immunogens, it is estimated that80-160 μg of virus particles will provide approximately the same amountof receptor antigen as 10⁷ cells (assuming 10⁶ copies of receptor percell, 10⁷ cells would contain 10¹³ receptors). Lipoparticles areestimated to carry 50-100 receptors/particle, and the MW of a retrovirusis approximately 4×10⁸ g/mol. So 80-160 μg of retrovirus is required todeliver 10¹³ receptors. This range of dosage is consistent withexpectations based on prior experience and comparison to viral vaccines(above). Because Lipoparticles are expected to deliver a higher qualityantigen (purified or concentrated and retaining structural integrity) ina better format for processing (particulate), a 100 μg lipoparticleprime is used and subsequently three 50 μg boosts (250 μg total) areused. A higher dosage of antigen is also used (550 μg total) in order toassess improved responsiveness with increased dosages. Specifically, onegroup of mice receives 250 μg of lipoparticles in prime immunization and100 μg in each of three boosts. The responsiveness of the sera of miceis tested for reactivity to determine the increase in responsivenessgained with this protocol. A second series of increased dosage (1 mgtotal) is employed if experimental results indicate that furtherimprovements could be gained.

The use of adjuvants is also tested. For these experiments, threedifferent conditions—no adjuvant, Ribi adjuvant, and TiterMaxadjuvant—are used to enhance the immune response to lipoparticles.Particles containing 100 μg CCR5 are mixed with an equal volume ofemulsified adjuvant for the priming immunization. The first boost alsocontains adjuvant, but subsequent boosts are administered withoutadjuvant. Seras are screened, as above, to determine the level ofreactivity increase under each condition. Seras are screened forblocking of Mabs to determine the epitope response of each condition andto ensure that no condition is producing Mabs only to linear epitopes.

Example 58 Generation of a Neutralizing Humoral Immune Response Using aLipoparticle as a Vaccine

The preceding examples have demonstrated that lipoparticles presentantigens for antibody production in a variety of ways, and that theseantibodies are useful for a variety of applications. In this examplelipoparticles are used for vaccination.

Open reading frame 74 (ORF74) is a constitutively active seventransmembrane (7TM) receptor stimulated by angiogenic chemokines, andinhibited by angiostatic chemokines. ORF74 is encoded by humanherpesvirus 8 (HHV8). Expression of ORF74 has been linked to developmentof rapidly growing, metastasizing, highly vascularized, tumors. Onemodel for the elimination of these tumors would be to generate an immuneresponse against cells expressing ORF74.

Since ORF74 is a 7TM receptor, and the protein relies on a lipid bilayerto maintain its structure, it is unlikely that vaccination with just theprotein would elicit the proper immune response. Killed or attenuatedHHV8 virus vaccination is not sufficient because the virus does notexpress the protein on its own membrane. The use of lipoparticles forvaccination offers the unique opportunity to present the ORF74 receptorin its transmembrane form and to use a particle similar in size andcomposition to a virus.

Successful vaccination against ORF74 would prevent the formation oftumors and prevent cancers developed from this pathway, by eliminatingcells which express this receptor (i.e. cells infected by HHV8 sinceonly HHV8 encodes this receptor).

Similarly, HER2 (human epidermal growth factor receptor 2) is a receptorfound to be over expressed in 25% to 30% of breast cancer patients. Thisover expression increases cell growth and division, making the cancermore aggressive than types not expressing HER2. Herceptin is a Mab whichis used as a drug to block HER2. Vaccination against HER2 may serve asimilar purpose—to slow the cancer, with the exception being that thebody would generate its own immune response, effectively immunizing thepatient against breast cancer.

Lipoparticles comprising either HER2 or ORF74 are administered to ananimal at a dosage of 100 ug of lipoparticles per 20 g of animal byinjecting intraperitoneal. Boosters of 100 ug lipoparticles per 20 ganimal are administered at 2 weeks and 4 weeks after the initialinoculation. Antibodies from the animal are monitored by FACS to measurethe immune response against ORF74 and HER2. Additional boosterinjections may be made to increase the titer of antibodies in the seraif desired. The animal is then challenged (e.g. with a tumor or withHHV8) in order to determine the protection given by the vaccination.

Example 59 Using a Lipoparticle and an Immunostimulating Component toImprove an Immune Response

Lipoparticles are used in conjunction with other immunomodulators, whichwill serve to optimize the potency and longevity of the immune response.The lipoparticle itself is also improved to increase the humoral immuneresponse.

Recent evidence suggests that using multiple vaccine vehicles to presenta common antigen in a sequenced prime-boost protocol may serve to betterinduce a humoral immune response. The secondary immunogen can be addedin several ways including 1) as a protein co-injection, 2) in cellsexpressing the protein, 3) as a killed or attenuated virus, or 4) byseveral genetic techniques whereby the DNA in introduced to the antigenpresenting cells (APCs) via plasmid DNA vehicles or a variety of viralvectors. For the case of an integral membrane protein which is notpresent on a normal virus, any of these techniques except the killed orattenuated virus (#3) could be employed.

For example, lipoparticles containing ORF74 are co-injected with plasmidDNA (e.g. pcDNA3 expressing ORF74 under a CMV promoter) vehicles or avariety of viral vectors (e.g. a recombinant adenovirus expressing ORF74under a CMV promoter) to increase the immune response. Alternatively, alipoparticle comprising ORF74 is coadministered with IL-2. IL2 normallyserves to signal CD4+ immune cells to divide. IL2 administration isalready in use clinically for some cancer patients and it is beingtested for use in AIDS patients. It is the CD4+ cells which die duringan HIV infection and IL2 administration has been used to boost thesecells to maintain an immune response. In the case of vaccines, IL2co-administration may increase the number of CD4+ cells beyond thenormal amount and improve the immune response to the immunogen beingpresented by the lipoparticle. IL2 use has been demonstrated to improvethe immune response from a peptide vaccination.

Example 60 Making Antibodies Using Lipoparticles and Macrophages

Lipoparticles can also be modified to mimic the effects of membraneproteins and antigens which are present in the cells of the immunesystem. Co-injection of this type of particle may serve to boostindividual steps of the immune response and improve the overallresponse.

A lipoparticle which has on its surface an antigen bound to MHC class 1may mimic the effects of a macrophage which is presenting an antigen,and stimulate a cytotoxic T-cell to activate. To generate thislipoparticle, macrophages are harvested from a mouse that has beenimmunized with a desired immunogen. Once the macrophages begin topresent the antigen, lipoparticles are generated from the macrophagesfollowing the methods described herein and in U.S. Patent Application US2002/0183247A1, U.S. Ser. No. 60/491,477, filed Jul. 30, 2003, and U.S.Ser. No. 60/491,633, filed Jul. 30, 2003. These particles should containall the membrane proteins of the macrophage and can then be used as avaccine. Lipoparticles can also be engineered to express MHC class Imembrane protein. Alternatively, lipoparticles can be engineered thatexpress the antigen and MHC class II. Lipoparticles can alternatively bebathed in MHC proteins to elicit binding prior to immunization. Thiscomplex may bind to an activated helper T-cell, stimulating thetransformation of the relevant B cell into an antibody-secreting plasmacell.

The experiments for this example will proceed as in described hereinwith the addition of secondary immunogens or modifications to theLipoparticle.

Example 61 Use of Libraries to Isolate Monoclonal Antibodies

Previous examples have relied upon an organism, such as a mouse orhuman, to generate antibodies against an injected lipoparticle. In thisexample a library of human Monoclonal antibodies is expressed in Phageor using Ribosome display and screened using lipoparticles expressing aprotein of interest. In this manner, a monoclonal antibody to theprotein of interest will be isolated ex vivo. This avoids the problemsof an antigen not being immunogenic or of an immune response directed tothe Gag protein of the lipoparticle. Phage libraries have been generatedby others to express human antibody fragments, and can contain >10⁷unique antibodies. The combination of this library and lipoparticleswill provide a powerful tool for rapid antibody isolation.

lipoparticles containing CCR5 are constructed and bound to high bindingELISA plate wells by incubating particles in Hepes Buffered Saline (HBS,10 mM Hepes pH 7.5, 150 mM NaCl) buffer for 2 hours. The unboundlipoparticles are washed away with three washes of HBS. Non-specificbinding sites are blocked using HBS containing 3% BSA. A commercialphage library of human Mab is incubated with the lipoparticles. Unboundphage are washed away by washing wells three times with HBS. Bound phageare eluted by washing with 100 mM triethylamine, pH 11. The eluted phageare neutralized in 1M Tris-HCl, pH 7.4. The resulting phage areamplified in bacteria. The procedure above can be repeated three timesto isolate phage that are specific to the membrane protein of interest.Isolation of a plaque of phage results in the identification of a singleMab which binds to CCR5. One skilled in the art would recognize thatphage panning could also be conducted using whole virus or virus-likeparticles.

Alternatively, a method that avoids the use of bacteria and phage butaccomplishes similar results is the use of ribosome display. This issimilar to using a phage library but instead the antibodies to bescreened are attached to a polysome, as described in He M, and TaussigM. Briefings in functional genomics and proteomics. Vol 1. no 2.204-212. July 2002. Once isolated, the mRNA in the polysome is amplifiedfor the identification of the antibody.

Example 62 Transfection of CCR5 into 293T Cells Using a Lipoparticle

This experiment involves the transfection of the CCR5 chemokine receptorpresent in the membrane bilayer of CCR5 lipoparticles into the targetplasma membrane of 293T cells. The transfection of CCR5 can be verifiedusing a Calcium Flux assay. A flux in the intracellular calcium oflevels of the transfected target cell is expected when stimulated withthe agonist CCR5 ligand MIP1-α.

Approximately 3×10⁶ 293T cells are cultured in a 60 mm plate. 20 μl ofthe Pro-Ject™ reagent (Pierce Biotechnology, Inc., Rockford, Ill.),which is obtained as a thin film of powder on a tube that is thendissolved by adding 250 ul methanol and is pipetted to the bottom of anEppendorf™ tube and dried after allowing 1-2 hours for evaporation. In aseparate tube, 4 μl of CCR5 lipoparticles (6.5×10⁷ particles/μl),prepared as previously described (Hoffman, et al. (2000), Proc. Natl.Acad. Sci. USA, 97:11215-11220), is diluted in 250 μl Hepes BufferedSaline (HBS). The lipoparticle/HBS mixture is added to the Eppendorftube containing the dried Pro-Ject™ reagent and is mixed by pipetting upand down. The lipoparticle/Pro-Ject™ mixture is vortexed for threeseconds and incubated 3-5 minutes at room temperature. 2.2 ml Optimem™is added to the lipoparticle/Pro-Ject™ mixture. The existing 293T mediais aspirated and replaced with 2.5 ml of thelipoparticle/Pro-Ject™/Optimem mixture and subsequently incubated for 4hours at 37° C.

To commence the calcium flux assay, 25 μl of Fura-2AM (1 mM in DMSO) isadded to 10 ml 10% FBS DMEM for a final concentration of 2.5 μM. Theexisting 293T media is replaced with 2.5 ml of 2.5 μM Fura-2AM/mediamixture. The cells are then incubated for 1 hour at 37° C. Afterincubation, the cells are washed with PBS and incubated for 5 minutes inPBS to lift the cells. The cells are then centrifuged for 5 minutes andresuspended in 1 mM Ca/Mg PBS with 2% FBS at 2×10⁶ cells/ml.

3×10⁶ cells (1.5 ml) are used per Ca Flux reaction. A 5 μl MIP1-α (0.1μg/μl) injection is used to challenge the 293T target cells and detect aCa Flux, as previously described (Doranz, et al. (1999), J. Virol.,73:2752-2761), by placing cells in a 4.5 ml cuvette, stirring at lowspeed, and injecting reagents at desired times.

Since the 293T cell line does not endogenously express CCR5 on theplasma membrane, the transfection of CCR5 into the target 293T cellmembrane bilayer can enable the cell to respond to an external MIP1-αligand challenge. Intracellular calcium levels can increase five- toseven-fold from basal state when the cell is challenged with MIP1-α.When loaded with Fura-2AM, the cell can fluoresce during this flux andallow proper detection of CCR5 transfection.

Example 63 Detection of Fusing Cells Using a Lipoparticle

This experiment involves the detection of two cells fusing, which can beused to detect the interaction of a membrane protein with its ligand,which can also be a membrane protein. The CCR5 chemokine receptor,present in the membrane bilayer of CCR5 lipoparticles, is used totransfect the target plasma membrane of quail QT6 cells. Thetransfection of CCR5 can be verified using a cell fusion assay with aluciferase reporter, as previously described (Doranz, et al. (1996),Cell, 85:1149-1158). Additionally, the confirmation of a proteininteraction can also be confirmed when the resulting interaction causesthe cells to fuse together. Briefly, HeLa cells are prepared as effectorcells and QT6 cells are prepared as target cells. A fusion event betweeneffector cells and target cells, as detected by luciferase activity,indicates the transfection of CCR5 into target QT6 cells.

For effector cell preparation, 2×10⁵ (a 24-well well) HeLa cells arecultured and prepared for vaccinia infection. Vaccinia viruses vTF7-3(expresses T7-polymerase) and vBD3 (expresses HIV-1 Envelope protein89.6) are used to infect HeLa cells at a Multiplicity of Infection (MOI)of 10 each. Vaccinia strains are prepared by incubating virus with 1volume of 0.25 mg/ml trypsin for 30 minutes while vortexing every 5-10minutes. Once thawed and trypsinized, the virus is added to the effectorcells and incubated for 2 hours in half volume at 37° C. After two hoursof infection, the culture media is replaced with 10% FBS mediacontaining rifampicin (1× final concentration) and allowed to incubateovernight at 32° C. When ready for use, Effector cells are washed withPBS, centrifuged for 5 minutes, and resuspended at 2×10⁶ cells/ml in 10%FBS media containing AraC and rifampicin.

For target cell preparation, 2×10⁵ QT6 target cells are transfected bycalcium phosphate. Precipitate is prepared by combining 17.5 μl sterilewater, 2.5 2M CaCl₂, 0.5 μl CD4-PT4 plasmid (1.385 μg/μl), 0.7 μlT7-luciferase plasmid (0.9 μg/μl), and 0.7 μl PHF-GFP plasmid (0.94μg/μl). Plasmids can be prepared as previously described (Doranz, et al.(1996), Cell, 85:1149-1158). The plasmid mixture was subsequently addedto 20 μl 2×HBS while vortexing to ensure adequate mixing and precipitateformation. The precipitate is subsequently incubated for 15 minutes atroom temperature and then added to existing QT6 media. QT6 cells areincubated with the calcium phosphate precipitate for 4-6 hours. Cellmedia is subsequently replaced with 10% FBS DMEM, and cells areincubated overnight at 37 C.

To begin the CCR5 transfection process, 2.5 μl of the Pro-Ject™ reagentdissolved in methanol is pipetted to bottom of an Eppendorf tube andincubated for 1-2 hours to permit methanol evaporation. In a separatetube, 4 μl of CCR5 lipoparticles (6.5×10⁷ particles/μl and prepared asdescribed herein) are diluted in 10 μl Hepes Buffered Saline (HBS). Oncecombined, the lipoparticle/HBS mixture is added to the tube containingdried Pro-Ject™ reagent and thoroughly mixed by pipetting up and down.The lipoparticle/Pro-Ject™ mixture is briefly vortexed and incubated for3 to 5 minutes at room temperature. After the brief incubation, Optimemserum-free media is added to the lipoparticle/Pro-Ject™ for a finalvolume of 250 μl. Existing QT6 media is replaced withlipoparticle/Pro-Ject™ mixture and incubated for 4 hours at 37° C.

To initiate the fusion event, 100 μl of effector HeLa cells (2×10⁵cells) are added to 2×10⁵ target QT6 cells and incubated for 8 hours at37 C. After incubation, cell lysate is prepared by adding 200 μl of PBSwith 1% Triton X-100 and incubating at room temperature for one minute.Luciferase activity is measured by adding 50 μl of cell lysate to a96-well plate with 50 μl Luciferase substrate (Promega) per well.Luciferase activity is detected using an ML3000 luminometer.

The QT6 cell line does not endogenously express CCR5 on the plasmamembrane. The transfection of CCR5 into the target QT6 plasma membranebilayer can enable the HeLa effector cells expressing the 89.6 HIV-1envelope protein to fuse with the QT6 target cells expressing both CD4and CCR5. The fusion event permits the effector's T7-polymerase totranscribe the T7-driven luciferase construct present in the targetcells. Successful transfection of CCR5 can allow a fusion event betweenthe effector and target cells with commensurate detection of luciferaseactivity.

Example 64 Transfection of Membrane Proteins Toxic to a Cell

The ion channel Kv1.3 can be toxic when over-expressed in some celltypes. Nevertheless, Kv1.3 is a membrane protein of considerableinterest. Kv1.3 can be incorporated into lipoparticles using theprotocols described in US 2002/0183247A1, U.S. Application Ser. No.60/491,477, U.S. Application Ser. No. 60/491,633, and U.S. ProvisionalSer. No. 60/498,755, filed Aug. 29, 2003. Alternative cell types can beused for production of lipoparticles in which Kv1.3 is less toxic. Kv1.3can then be transfected into an NT2 differentiated neuron, or anothercell type, using the membrane protein transfection protocol describedabove. Membrane transfection enables mutagenic study by circumventingpotential complications of DNA transfection.

Example 65 Transfection of a Mutant Membrane Protein

Mutagenesis of chemokine receptor CCR5 provides an effective way tostudy its interaction with HIV-1 and chemokines. However, CCR5 cannotalways be introduced into desired cell types, such as differentiatedmacrophages that lack endogenous CCR5 (CCR5-delta32 homozygotes). Theintroduction of mutant forms of CCR5 would enable the study of theprotein and its functional properties within an important cell type.Mutants of CCR5 can be incorporated into lipoparticles using theprotocols described US 2002/0183247A1, U.S. Application Ser. No.60/491,477, and U.S. Application Ser. No. 60/491,633. Point mutations,chimeras, and truncation of CCR5, such as previously described (Doranz,et al. (1997), J. Virol., 71:6305-6314, Rucker, et al. (1996), Cell,87:437-446), can be made. CCR5 mutants are then transfected into primarymacrophages. Some of the macrophages can lack endogenous CCR5, oranother cell type, using the membrane protein transfection protocoldescribed in Example 62. Once within the cell, the transfected proteincan be used to study signaling pathways within this cell.

Example 66 Transfection of a Membrane Protein with AlteredPost-Translational Modifications

Post-translational modifications of the chemokine receptor CCR5 areimportant in regulating its location and signaling properties within acell. However, post-translational modifications of CCR5 cannot always beintroduced as desired. The control of post-translational variants ofCCR5 would enable the study of the protein and its functionalproperties. CCR5 can be incorporated into lipoparticles as describedherein. To control the post-translational state of CCR5, lipoparticlesare biochemically manipulated to introduce or eliminatepost-translational modifications. The lipoparticle is treated withendoglycosidase F to remove N-linked carbohydrates. For otherextracellular modifications, lipoparticles are treated with chemicals,enzymes, or reagents in order to modify N-linked carbohydrates, O-linkedcarbohydrates, or sulfated residues. The lipoparticle are made tocontain a phosphatase to remove any phosphorylation sites added to CCR5.For other intracellular modifications, the lipoparticle interior is madeto contain chemicals, enzymes, or reagents that can function as kinases,phosphatases, or intracellular binding partners. Thepost-translationally modified CCR5 protein is then transfected into a3T3 cell, or another cell type, using the membrane protein transfectionprotocol described in Example 62. Once within the cell, the modifiedprotein can be used to study signaling pathways within this cell.

Example 67 Membrane Protein Transfection for Delivery of a PoorlyExpressed Membrane Protein

Some membrane proteins are not efficiently transcribed, translated, orprocessed within a cell, resulting in poor surface expression. In othercases, the membrane protein is quickly internalized, degraded, orinactivated, also resulting in poor surface expression. Membrane proteintransfection can overcome these obstacles by delivering high levels ofthe membrane protein directly to the surface of a desired cell. Thechemokine receptor CCR3, for example, is often poorly expressed on thecell surface. CCR3 is incorporated into lipoparticles using theprotocols described in US 2002/0183247A1, U.S. Application Ser. No.60/491,477, and U.S. Application Ser. No. 60/491,633. CCR3 istransfected into 293 cells, or another cell type, using the membraneprotein transfection protocol described herein. Once within the cell,the protein is used to study signaling pathways within this cell.

Example 68 Membrane Protein Transfection for Delivery of a TherapeuticMembrane Protein

The Cystic Fibrosis Transmembrane Regulator (CFTR) membrane protein is achloride channel that, when defective, is responsible for causing CysticFibrosis. Studies suggest that effective treatment of Cystic Fibrosiscould be achieved by administering the CFTR gene via gene therapy.However, gene therapy techniques, in which the DNA encoding a protein isdelivered to a target cell for therapeutic purposes, face manychallenges. The method disclosed herein, as described, for example, inExample 62 provides an alternative means of delivering the membraneprotein and providing an effective treatment to the disease. CFTR hasbeen incorporated into lipoparticles, using techniques previouslydescribed (Hoffman, et al. (2000), Proc. Natl. Acad. Sci. USA,97:11215-11220)). The lipoparticles are prepared as a pharmaceuticalcarrier or diluent. The lipoparticles are administered to theindividual. The delivery of the CFTR protein to effected tissue (e.g.lung) lacking a functional CFTR protein, using methods disclosed below,can ameliorate the disease. The cells that the membrane protein is to betransfected into are targeted. Method of targeting cells in vivo arewell know in the art, including, but are not limited, using an antibody,using a specific receptor on the surface of cell, and the like.

Example 69 Membrane Protein Transfection for Targeting of Stem Cells InVivo

A current limitation of stem cell therapies is the inability to localizetherapeutic stem cells to the appropriate target tissue. The chemokinereceptor CXCR4 is known to help regulate migration of hematopoietic stemcells in vivo (Lee, et al. (1998), Stem Cells, 16:79-88, Peled, et al.(1999), Science, 283:845-848). Membrane transfection of CXCR4 onto thestem cell plasma membrane would aid in the targeting of the stem cell tothe appropriate tissue type. CXCR4 lipoparticles are prepared accordingto the methods described in US 2002/0183247A1, U.S. Application Ser. No.60/491,477, and U.S. Application Ser. No. 60/491,633 and transfectedinto stem cells ex vivo (i.e. in a tissue culture dish before beingreintroduced into a patient) according to examples herein. The stemcells are then administered to a patient using methods known to those ofordinary skill in the art (Hui (2002), Technol Cancer Res Treat,1:373-84).

Example 70 Membrane Protein Transfection in a Terminally DifferentiatedCell

DNA transfection of many cell types requires cell division in order toefficiently express the DNA of interest. Transfection using a viraldelivery vehicle also usually requires a dividing cell in order toefficiently express the DNA in the viral vector. The introduction ofmembrane proteins using membrane protein transfection withlipoparticles, however, has no such requirements. Many cell types, suchas differentiated cells and quiescent cells, are not efficientlytransfected using DNA transfection or viral delivery vehicles. Ectopicintroduction of membrane proteins via membrane protein transfectionprovides a suitable alternative to study protein interactions in saidcells. Lipoparticles comprising CCR5 are fused with the terminallydifferentiated neuronal cell line N-tera 2 according to the methodsdescribed herein. The transfected cell line is used to study thefunction of CCR5 in a non-dividing cell. One of ordinary skill in theart would recognize that other cells in a quiescent state could also betransfected in such a manner.

Example 71 Imaging and Quantifying Lipoparticles

To visualize and quantify lipoparticle concentration we have used afluorescent molecule that partitions into lipid environments and becomesfluorescent only when in a lipid environment. The membrane potentialprobe di-4-ANEPPS was obtained (Molecular Probes) as a powder,resuspended in 50% ethanol and 50% DMSO, and 1 μl was added to 100 μl ofa 1:100 dilution of lipoparticles in 0.22 um filtered HBS. Unlikewater-soluble dyes, di-4-ANEPPS is lipophilic, simplifying theincorporation and use of the dye with lipoparticles. The dye partitionedinto membranes nearly instantaneously and dye that does not partitioninto the membrane is non-fluorescent. The particles were then placedonto a hemocytometer under a cover slip. The hemocytometer was placed onthe stage of a fluorescence microscope and the lipoparticles werecounted using an oil-immersion 100× lens (Nikon) under redepifluorescent illumination. The lipoparticles could be imaged underepifluorescent illumination as small coronas of fluorescent light, witheach corona representing a single lipoparticle (data not shown). Thisnumber was mathematically extrapolated to lipoparticles per μl ofsolution by multiplying the number of fluorescent lipoparticles within adefined region of the hemocytometer by the volume of the hemocytometerthat that region represents. The formula for this calculation was:((Average #Lipoparticles per area visualized)*(1/0.0025 areavisualized)*(0.1 μl per area visualized)*(Dilution of Lipoparticles)).

One skilled in the art would recognize that other lipophilic dyes couldalso be used to observe lipoparticles. In fact, any other dye that haslipophilic properties could be used for the same purpose. Manylipophilic dyes do not fluoresce or fluoresce only weakly in an aqueousenvironment, meaning that the dye need not be separated from thelipoparticle in order to visualize the lipoparticle. di-4-ANEPPS, diI,diBAC4, and Nile Red have been used to detect lipoparticles.

Example 72 Incorporation of Fluorescent Reporter Proteins intoLipoparticles

In order to detect and quantify lipoparticles in a more efficient andreproducible manner, we created a Gag protein with an Enhanced GreenFluorescent Protein (GFP) fused to the C-terminus of Gag. GFP was clonedusing PCR to fuse the GFP protein to the C-terminus (amino acid 533) ofGag (nucleocapsid protein). When the construct is transfected intocells, a Gag-GFP fusion protein is produced and drives the budding ofretroviral particles. We have used this construct to producelipoparticles that, when visualized with a 100× lens underepifluorescent illumination, are readily visible (data not shown).

Lipoparticles containing receptors fused to a fluorescent reportermolecule (CXCR4-GFP and CCR5-GFP) were also produced. Both membraneproteins are G-protein coupled receptors with GFP fused to theC-terminus (intracellular) of the GPCR. Both of these lipoparticles wereproduced and used for imaging and quantification using microscopevisualization.

Example 73 Protein Concentration of Lipoparticles

The overall protein concentration of the lipoparticle preparation wasdetermined using a BCA assay kit (Pierce) and comparing to a knownquantity of purified lipoparticles. 5 μl of lipoparticles were placedinto a well of a microplate. 200 μl of BCA working reagent was added toeach well, mixed on a shaker for 30 sec and incubated at 37° C. for onehour. The plate was cooled to room temperature and the absorbance at 562nm was measured using an absorbance detector to determine proteinconcentration of each well of the microplate. When the experiment wasconducted with protein standards of known concentration, the proteinconcentration of the lipoparticle preparation could be determined (Table14). Alternative protein concentration kits have also been used,including microBCA and NanoOrange (data not shown).

TABLE 14 Protein Concentration of lipoparticle preparation Abs @ 570nm - Conc. Conc. Sample Blank Abs @ 570 nm (μg/ml) (mg/ml) INT- 1.241829 0.829 0037A INT- 0.49 292 0.292 0038A

Example 74 Dynamic Light Scattering

The size and purity of a lipoparticle preparation was determined usingDynamic Light Scattering (DLS). To determine the size distribution ofthe lipoparticles in a purified population of lipoparticles,approximately 2 ng of purified lipoparticles were suspended in 35 μl ofHepes Buffered Saline (HBS) in a microcuvette. The sample was placed ina Proterion DynaPro Dynamic Light Scatter machine and counts weremeasured. The data was analyzed by displaying the counted population ona histogram to determine the size distribution on the X-axis and theintensity of measurement on the Y-axis (FIG. 15). DLS is a measurementof both lipoparticle size (diameter) and lipoparticle purity (breadth ofpeak and number of peaks). A pure population is represented by arelatively narrow peak on the histogram whereas a less-pure preparationresults in numerous peaks or a very broad peak. Polydispersity is ameasure of the breadth of a DLS peak, and a value of <20% is generallyconsidered a homogeneous peak. The size of the lipoparticle wasdetermined to be 207.4 nm in diameter with a polydispersity of 18.1%.200 nm beads (diameter as specified by the manufacturer) were used as acontrol (bottom panel) and measured 235.9 nm in diameter with apolydispersity of 8.3%.

Example 75 Quantification of Lipoparticles

In order to quantify the number of lipoparticles in a sample, we usedthree methods: fluorescent imaging, dynamic light scattering, andspectroscopy correlation. To validate the accuracy of these methods wefirst tested 200 nm synthetic fluorescent beads (YG Fluoresbrite beads,Polysciences) using these three methods. The beads were imaged andcounted by microscopy using a 100× lens and epifluorescent illuminationin a calibrated hemocytometer. The beads were also subjected to dynamiclight scattering using a Proterion DynaPro instrument. Finally, thebeads were placed in a cuvette in a Perkin-Elmer LS50B fluorometer andlight scatter was measured with an excitation of 540 nm, an emission of570 nm. On skilled in the art would recognize that other wavelengthscould also be used to measure light scatter, and that the wavelengthwill be a function of the size of the suspended material being measured.All methods were then plotted against the bead concentration as given bythe manufacturer and measured during production by weight (FIG. 15C).All measurements were proportional to each other and to the publishedbead count, with some variation due to dilution accuracy.

We then used the same techniques to measure the concentration oflipoparticles in a given sample. The lipoparticles tested contained theGag-GFP fusion protein, thus enabling easy visualization. Thelipoparticles were imaged and counted by microscopy using a 100× lensand epifluorescent illumination in a calibrated hemocytometer. Thelipoparticles were also subjected to dynamic light scattering using aProterion DynaPro instrument. 200 nm Fluoresbrite beads were alsoincluded in this experiment for comparison. The results demonstratedthat the measurements were directly correlated over a largeconcentration range. Furthermore, the intensity of DLS could be used topredict the concentration of lipoparticles (particles/ul) using a simpleequation (FIG. 15D).

Example 76 Quantification of Receptors Using Western Blot

The number of receptors per lipoparticle is a measurement that can beused in determining the efficacy of membrane protein incorporation intothe lipoparticles. Western blot is one technique that can be used toquantify the amount of specific membrane protein present in a sample.10E9 lipoparticles containing two V5 epitope tagged GPCRs (CCR5 andCXCR4) were run on a 12% acrylamide SDS-PAGE gel. Predetermined amounts(30, 100, and 300 ng) of a control protein (GFP-V5) were run in separatelanes of the gel. The control protein consists of purified andquantified green fluorescent protein (GFP) that contains the same V5epitope tag. The gel was transferred to PVDF and the membrane wasincubated with an antibody against the V5 epitope tag. Finally, asecondary antibody conjugated to horseradish peroxidase (HRP) was usedto detect the primary antibody. A chemiluminescent reagent (PierceFemtoSignal) was added and the blot was visualized using anAlphaInnotech Fluorchem 8900 (FIG. 15E). Quantification withAlphaInnotech AlphaEase software allowed quantification of the amount ofV5 tag detected in the lipoparticle lane and comparison to the standardscontaining the same V5 tag of known quantity in the other lanes.

One skilled in the art would recognize that any epitope tag could beused to achieve similar quantification results. One skilled in the artwould recognize that other proteins, including Gag, could also bequantified in a similar manner. SDS-PAGE and Western blot also allowedvisualization of the quality of the receptor (e.g. degraded,full-length, glycosylated, dimers, etc.). The standard protein containsa known quantity (μg) of protein, which could then be converted to#moles and #molecules. By using these standards, the estimated number ofμg, moles, and molecules of receptor protein was determined in thelipoparticle sample. 10 picomoles of CXCR4 and 4.9 picomoles of CCR5were incorporated in 10E9 particles.

Example 77 Quantification of Receptors Using Sypro Staining

Lipoparticles containing the GPCR CXCR4 were purified using Ni+2 beadsas follows: 1.8×10¹¹ particles (2.4 mL of 0.33 mg/mL total protein) werelysed in an imidazole lysis buffer solution comprised of 20 mM Tris pH7.5, 150 mM NaCl, 1% Triton X-100, 20% glycerol and 20 mM imidazole(final concentrations are after mixing with the particle solution, finalvolume was 4 mL). The solution was vortexed and then incubated at 4° C.with rocking for 20 minutes. 25 uL of Ni-NTA His-Bind Superflow beads(Qiagen) were added and the solution incubated with rocking overnight at4° C. The beads were pelleted by gentle centrifugation in a tabletopcentrifuge (5 minutes at 2,000 RPM), resuspended in 100 uL of the lysisbuffer, and transferred to an eppendorf tube. The wash step was repeatedand the beads were washed with increasing concentrations of imidazole inthe lysis buffer from 20-100 mM imidazole (2×100 uL wash for 20, 50, 75and 100 mM imidazole buffers). CXCR4 was eluted from the beads withlysis buffer containing 250 mM imidazole in two 100 uL fractions.Residually bound lipoparticles were eluted with lysis buffer containing500 mM imidazole in two 100 uL fractions.

Samples from the purification were run on SDS-PAGE (4-20% acrylamidegradient) and the gel stained using Sypro Orange (Molecular Probes). 30uL of the following samples from the purification were run: startingmaterial (unpurified lysed lipoparticles), flow through, 20 mM imidazolewash, elution fractions from 50-500 mM imidazole (FIG. 15F). Inaddition, a similar but separate SDS-PAGE gel was run, stained withSypro Orange, and quantified. 30 uL of the following samples from thepurification were run on this second gel: starting material (unpurifiedlysed lipoparticles), 250 mM imidazole elute fractions 1 and 2 (100 uLtotal volume each), 500 mM imidazole elute fractions 1 and 2 (100 uLtotal volume each). In addition 30, 100 and 200 ng samples of purifiedhPRR2 (Geraghty et al Science. 1998 Jun. 5; 280(5369):1618-20) were runas standards.

To calculate the total amount of CXCR4 in the preparation, the hPRR2bands (background subtracted) were quantified using AlphEaseFC (AlphaInnotech) and the data plotted for a standard curve. The CXCR4 band ineach of the elution fraction lanes was quantified and the values used tocalculate the amount of protein in each band. The total amount of CXCR4in the preparation was back-calculated from these values. To calculatethe percent of total protein of CXCR4, the entire lane of the startingmaterial was quantified using AlphaEaseFC as above. The CXCR4 band inthat lane was quantified and the percent of total calculated. Oneskilled in the art would also recognize that the amount of protein inthe purified CXCR4 sample could also be quantified by a total proteinconcentration analysis such as a BCA assay.

Example 78 Quantification of Receptors Using Dot Blot

A dot blot was performed to quantify the amount of receptor in alipoparticle sample. Dilutions of a purified protein standard, a GFPprotein containing a V5 epitope tag, were included on the same dot blot.Dilutions of two sample lipoparticle preparations containing tworeceptors, CXCR3 and CD4, with the same V5 tag were also included. Thesamples were blotted through a 96-well manifold onto nitrocellulose andthen probed with an anti-V5 antibody. The dot blot was imaged using anAlpha Innotech Fluorchem and spots were quantified and compared. Acalibration curve was constructed from the standard protein curve andcurves were also constructed from the lipoparticle samples with unknownreceptor quantity (FIG. 15G). Using the calibration curve, we were ableto estimate the amount of CXCR3 and CD4 in these two lipoparticle prepson a ug/ul basis.

Example 79 Quantification of Receptors Using Ligand Binding

Radioligand binding curves were performed to detect ligand binding tolipoparticles and to estimate the concentration and receptors.Radiolabeled SDF-1α (Perkin-Elmer) was used to bind lipoparticlescontaining the CXCR4 membrane protein. Lipoparticles were resuspended ina total of 100 ul of Hepes⁺⁺ Binding Buffer (50 mM Hepes 7.4, 5 mMMgCl₂, 1 mM CaCl₂, 100 mM NaCl, and 2% BSA) together with 0.1 nMradioligand. Increasing amounts of cold ligand (Peprotech) were alsoincluded where indicated. The mixture was incubated 1 h at roomtemperature and then filtered through Whatman GF/C filters soaked in0.2% polyethyleneimine (PEI). Filters were counted in a Wallac gammacounter. The results indicated that CXCR4 on the lipoparticles wasstructurally intact and capable of binding ligand (FIG. 15H). Theresults also demonstrated a very high concentration of CXCR4 in thepreparation, 230.2 pmol/mg. A titration curve using increasing amountsof lipoparticles also indicated very high concentrations of CXCR4, withan EC50 of 0.15 ug (FIG. 15I). These numbers were used to calculate thetotal amount of receptor per unit volume and per unit lipoparticle.

Example 80 Detection of Membrane Protein Structural Integrity UsingVirus-Detection ELISA

To determine the structural integrity of membrane proteins incorporatedinto Lipoparticles, we have utilized a technique, termed Virus-DetectionELISA, (VELISA), that allows quantification of the structural integrityof a membrane protein within the lipoparticle. VELISA is a modifiedenzyme linked immunosorbant assay (ELISA). VELISA has been performed onboth purified and unpurified lipoparticles. Lipoparticles containingeither CCR5 or CXCR4 were used in a VELISA assay by bindinglipoparticles to ELISA wells coated with antibodies against either CXCR4or CCR5. Briefly, 0.75 μg of a primary monoclonal antibody was adsorbedto each well of a high-binding ELISA plate. The wells were incubatedovernight, washed with HBS and blocked using 3% BSA in HBS. Next, 0.5 μgof purified lipoparticles were added to each well, and the plates werecentrifuged for 1 hour at 3,000 rpm to sediment lipoparticles to thebottom of the wells. The wells were washed three times with HBS.Subsequently, 200 μl of 1% sodium dodecyl sulfate (SDS) was added toeach well and incubated at room temperature for 10-60 minutes, followedby vigorous mixing. 100 μl of lipoparticle lysate was transferred to adot-blot apparatus for detection of Gag protein using anti-Gag rabbitsera and anti-rabbit secondary antibody conjugated to horseradishperoxidase. The Gag protein is a structural protein that can be detectedin order to quantify the amount of lipoparticles bound during VELISAassay. Specific binding of the lipoparticle during the VELISA assay ismediated solely by the interaction of a membrane protein with anantibody that recognizes that membrane protein (FIG. 16). Detection ofGag indicates that the membrane protein was embedded in the lipoparticleformed by the Gag protein. Control lipoparticles and antibodies are usedto indicate specificity. A similar VELISA assay was performed in thepresence of various additives to test the effect of each additive on themembrane protein-antibody interaction. CXCR4-containing lipoparticleswere bound to an anti-CXCR4 MAb (447.12) or a non-specific anti-CCR5 MAb(45523) in the presence or absence of various additives (ethanol, DMSO,glycerol, sucrose, trehalose, Pluronics, polyethylene glycol, phosphatebuffered saline, tris buffered saline, or Triton X-100). In this case,ELISA, rather than a dot blot, was used to detect Gag after specificbinding by binding the lysate to a new ELISA plate and detecting boundprotein using a rabbit anti-Gag sera. Results demonstrate that mostadditives had little or no effect on binding (FIG. 16B). Additives suchas detergent, however, completely destroyed lipoparticle structure andbinding, as expected.

Example 81 Detection Membrane Protein Structural Integrity UsingAntibody-Detection Viral ELISA

To determine the structural integrity of membrane proteins incorporatedinto lipoparticles we have utilized another technique, termedAntibody-Detection Viral ELISA (AVELISA) that allows quantification ofthe structural integrity of a membrane protein within the lipoparticle.AVELISA is a modified enzyme linked immunosorbant assay (ELISA).Lipoparticles containing either CCR5 or CXCR4 were used in a AVELISAassay by binding lipoparticles directly to ELISA wells and thendetecting bound lipoparticles using antibodies specific for CXCR4 (12G5and 447.08) or CCR5 (CTC8). Briefly, 0.75 μg of purified lipoparticleswere adsorbed to each well of a high-binding ELISA plate. The plateswere centrifuged for 1 hour at 3,000 rpm to sediment particles to thebottom of the wells. The wells were washed with HBS and blocked using 3%BSA. Next, 0.5 μg of membrane protein-specific primary antibody wasadded to each well, and the plate was incubated for 2 h at roomtemperature. The wells were washed three times with HBS, and anHRP-conjugated secondary antibody was added to detect the primaryantibody bound to the membrane protein. The secondary antibody wasallowed to incubate for 30-60 min and then washed three times with HBS.The HRP was then detected with a chemiluminescent substrate for HRP(Pierce FemtoSignal reagent) in order to quantify the amount of antibodybound specifically to each membrane protein (data not shown). Controllipoparticles and antibodies are used in both configurations to indicatespecificity.

Example 82 Detection of Membrane Protein Structural Integrity UsingSensor Detection

In the present method, detection is accomplished in one of twoconfigurations. In the first, a lipoparticle is attached to the sensorsurface and a conformation-specific antibody is flowed across thesurface. Specific binding of the antibody to the membrane protein in thelipoparticle is indicative of the structural integrity of the membraneprotein. In the second configuration, the conformation-specific antibodyis attached to the sensor surface directly. Lipoparticles are thenflowed across the antibody surface and specific binding is detected whenthe membrane protein in the lipoparticle binds to the specific antibody.Control lipoparticles and antibodies are used in both configurations toindicate specificity. This assay is indicative of the structuralintegrity of the membrane protein within the lipoparticle and is used asa test to insure membrane protein integrity within lipoparticles.

Example 83 Detection of Membrane Protein Structural Integrity Using FlowCytometry

Another means of detecting the structural integrity of membrane proteinscontained in lipoparticles is flow cytometry. 1×10⁹ purifiedlipoparticles are labeled with a conformation-specificfluorescein-conjugated antibody (12G5) directed at the protein ofinterest (CXCR4). Control lipoparticles are prepared using no integralprotein or control proteins. In addition, control antibodies are used toconfirm specificity. The samples are analyzed on a FACScan flowcytometry measurement device. Data are analyzed using CellQuestsoftware. Comparing samples and control samples (no antibody,lipoparticles with no protein of interest) allows the structuralintegrity of membrane proteins on lipoparticles to be determined. Byusing a control (200 nm latex beads) containing a known number ofantibody binding sites, the number of receptors per lipoparticle canalso be determined.

Because flow cytometry measures individual events, flow cytometry canalso be used to quantify the number of lipoparticles in a given sample.The lipoparticles to be quantified can be made fluorescent using aGag-GFP fusion protein, a lipid dye, a receptor-GFP fusion protein, asecondary antibody bound to the lipoparticle, or any other method ofstaining a lipoparticle.

Lipoparticles can be attached to beads to facilitate detection by flowcytometry. The beads may be larger in size (e.g. 10 μm) in order tobetter accommodate a flow cytometer detector. In one embodiment, thebeads are fluorescently labeled. In another example, the lipoparticlesare biotinylated and the beads are coated with streptavidin tofacilitate linkage. In another example, the beads are coated with thelectin Wheat Germ Agglutinin (WGA).

Example 84 Detection of Membrane Protein Structural Integrity UsingImmunofluorescence

Another means of detecting the structural integrity of membrane proteinscontained in lipoparticles is by labeling the lipoparticles withfluorescently-conjugated antibodies or ligands and visualizing thelipoparticles. Lipoparticles and viruses have generally been thought tobe too small to stain and visualize in such a manner, but we disclosemethods herein to accomplish such staining. Briefly, 5×10⁶ CXCR4 or CCR5lipoparticles were incubated with 50 ng of primary antibody in a totalvolume of 10 μl HBS. Primary antibodies consist of specific andnon-specific antibodies (12G5 against CXCR4, 2D7 against CCR5). After 30minutes at room temperature, 90 μl of HBS was added to the lipoparticlesand the mixture was spun in an Eppendorf 5415c microfuge at 14,000 rpmfor 30 minutes at 4° C. The supernatant was removed and 10 μl ofCy3-anti mouse secondary antibody, diluted 1:400 in HBS, was added.After 30 minutes at room temperature, 90 μl of HBS was added and themixture was spun for 30 minutes as before. The supernatant was removedand the pellet was resuspended in 10 μl of HBS and visualized using anepifluorescent microscope. Lipoparticles were stained with antibodiesspecific to the receptor within the lipoparticles, but not withantibodies that did not react with receptors within the lipoparticles(data not shown). Alternative methods of detection, such as flowcytometry, could also have been used.

Example 85 Detection of a Lipoparticle

To quantify lipoparticle concentration we have used a fluorescentmolecule that partitions into lipid environments and becomes fluorescentonly when in a lipid environment. The membrane potential probedi-4-ANEPPS was obtained (Molecular Probes) as a powder, and resuspendedin 50% ethanol and 50% DMSO. A dilution of lipoparticles (0.1 ul to 10ul of purified lipoparticles, each in a total volume of 50 ul HBS) wasplaced into separate wells of a clear 96-well plate. Wells were eithercoated with WGA or left uncoated. Lipoparticles were spun for 1 h atroom temperature to adhere lipoparticles to the plate. The wells werethen washed with HBS and 50 ul of di-4-ANEPPS (diluted 1:100 in HBS) wasadded to each well. The fluorescence in each well was measured using amicroplate fluorometer (FIG. 17). Similar measurements were taken forpurified lipoparticle preparations without adhesion to the microplate(Table 15). Unlike water-soluble dyes, di-4-ANEPPS is lipophilic,simplifying the incorporation and use of the dye with lipoparticles. Thedye partitioned into membranes and dye that does not partition into themembrane is non-fluorescent. Therefore, fluorescence was measured onlywhen lipoparticles were present, and the amount of fluorescence wasproportional to the quantity of lipoparticles present. One skilled inthe art would recognize that other lipophilic dyes could also be used toquantify lipoparticles. In fact, any other dye that has lipophilicproperties could be used for the same purpose. Many lipophilic dyes donot fluoresce or fluoresce only weakly in an aqueous environment,meaning that the dye need not be separated from the lipoparticle inorder to quantify the lipoparticle.

TABLE 15 Lipoparticle Lot# Fluorescence 35A 2,432 34A 726 003-01-0005A456 32B 828 003-01-0005C 826 33A 1,529 99A 857 001-00-0002B 1,014006-01-0003A 3,554 002-00-0006A 230 006-00-0003A 879 002-07-0001B 1,138

Example 86 Determination of Receptor Purity and Concentration

The methods described in the examples above yield quantitativeinformation about the number and purity of lipoparticles, as well as thenumber and purity of membrane proteins within the lipoparticles. Each ofthese assays alone is valuable, but when used in combination, they areadditionally informative. For example, we have used the formulaX=(#Lipoparticles per μl)/(Total Protein Concentration) as an estimateof the purity of the lipoparticle preparation. The number oflipoparticles was derived from imaging and the Total ProteinConcentration was derived from BCA assay. The resulting calculation(Lipoparticles per ug total protein) is indicative of the purity of thelipoparticle preparation (Table 16). The higher the value the greaterthe purity of the preparation. A lower number may be indicative ofcontamination of the lipoparticle preparation with other proteins (e.g.serum or BSA).

TABLE 16 Lipoparticle purity and concentration. Particles/μl Protein [ ]Particles/ug Lot # Receptor (×E6) (ug/μl) (×E6) 1 CXCR4 7 0.40 18 2CXCR4 50 0.43 116

Similarly, we have used the formula X=(# Receptors perμl)/(#Lipoparticles per μl) as an estimate of the density of receptorswithin the lipoparticles. The number of lipoparticles was derived fromimaging and the concentration of receptors (in units of #molecules) wasderived from Western quantitation. The resulting calculation (Receptorsper Lipoparticle) is indicative of the density of receptors within thelipoparticle preparation. The formula calculates an average number ofreceptors per average lipoparticle, and individual lipoparticles maydeviate from this mean. The higher the value, the greater the density ofthe receptor within the preparation. A lower number may be indicative oflow incorporation of receptor into the lipoparticles (e.g. low receptorexpression, poor incorporation, etc.).

One skilled in the art would recognize that these calculations arevaluable not only for measuring lipoparticles, but also for measuringany particle (e.g. retrovirus, enveloped virus, virus, virus fragment,or virus derivative).

Example 87 Incorporation of Gαi into Lipoparticles by Fusion to Gag

A fusion protein comprising a G protein and Gαi was created by fusingthe Gα_(i2) isotype G protein to the Gag protein, at residue 1955 ofGag, using standard cloning methodology (see, for example, MolecularCloning: A Laboratory Manual 3^(rd) ed., Sambrook et al. Cold SpringHarbor Laboratory). Lipoparticles containing the Gag-G protein fusionand the GPCR CXCR3 were created by standard techniques. The presence ofG protein fusion proteins within producer cells and lipoparticles wasverified by Western blot using anti-G protein antibodies and anti-Gagantibodies (FIG. 19).

Example 88 Incorporation of G Proteins into Lipoparticles by Fusion toGag

A fusion protein comprising a G protein and Gag is created. The fusionprotein may also contain an amino acid linker, which allows adequatemobility of the G protein component for its interaction withincorporated GPCRs. The Gag-G protein fusion protein is created byfusing the Gα_(z) isotype G protein to the Gag protein, at residue 1955of Gag. Fusion proteins are created using standard cloning methodology(see, for example, Molecular Cloning: A Laboratory Manual 3^(rd) ed.,Sambrook et al. Cold Spring Harbor Laboratory) and in one embodimentincorporates a poly-alanine (15 residue) linker between the fusedprotein elements. Lipoparticles containing the Gag-G protein fusion andthe GPCR CXCR4 are created by standard techniques. The presence of Gprotein fusion proteins within producer cells and lipoparticles isverified by Western blot using anti-G protein antibodies and anti-Gagantibodies.

The ability of the G protein to interact with the GPCR is tested byimmunoprecipitation as follows: Lipoparticle membranes are disruptedusing 1.0% CHAPSO, and GPCRs are immunoprecipitated using an antibodyrecognizing an N-terminal HA epitope tag on the GPCR. Co-precipitationof G proteins with the GPCRs, as detected by Western blot, is indicativeof coupling. Interaction of the GPCR and G protein may also be confirmedby chemical cross-linking as follows: When added to mixtures, thecross-linking agent EGS (Pierce) covalently couples molecules that arein contact with each other (within 16 Å). The irreversible covalentcoupling achieved by this method would result in a shift in themolecular weight of the GPCR and co-localization of G protein asdetected by Western blot, indicating their functional association(proximity without specific interaction at this distance is atypical).Controls for both of these experiments may include pertussis toxininhibition of GPCR-G protein coupling, and non-GPCR membrane proteinswith the same HA epitope tag that do not interact with G proteins (e.g.CD4). One skilled in the art would recognize that incorporation ofadditional or alternative G protein isotypes, such as Gα_(i), Gα_(s),Gα_(q), and Gα₁₂, into lipoparticles can also be performed using this orsimilar techniques.

Example 89 Incorporation of G Proteins into Lipoparticles by Fusion toGPCRs

The Gα_(z) protein isotype is fused to the GPCR CXCR4 at the membraneprotein's C-terminus. The GPCR-G protein fusion protein is incorporatedinto lipoparticles using techniques previously described herein. Alinker and/or a protease cleavage site may be included, as desired, toprovide adequate spacing of the fusion proteins to allow interaction.The presence of GPCR-G protein fusion proteins within producer cells andlipoparticles is verified by Western blot using anti-G proteinantibodies and anti-CXCR4 antibodies. One skilled in the art wouldrecognize that additional or alternative G protein isotypes andadditional or alternative GPCRs could be fused and incorporated intolipoparticles in a similar manner.

Example 90 Incorporation of G Proteins into Lipoparticles by Fusion toan Inert Membrane Protein Anchor

The Gα_(z) protein isotype is fused to the C-terminus (cytoplasmic) ofthe single transmembrane protein CD4. Alternatively, a fusion proteincontaining a truncated version of CD4 with a shorter C-terminus couldalso be constructed. The CD4-G protein fusion protein is incorporatedinto lipoparticles using techniques described herein. A linker and/or aprotease cleavage site may be included, as desired, to provide adequatespacing of the fusion proteins to allow interaction. The presence ofCD4-G protein fusion proteins within producer cells and lipoparticles isverified by Western blot using anti-G protein antibodies and anti-CD4antibodies. One skilled in the art would recognize that additional oralternative G protein isotypes and additional or alternative membraneproteins could be fused and incorporated into lipoparticles in a similarmanner.

Example 91 Incorporation of G Proteins into Lipoparticles UsingTransient Poration

Lipoparticles incorporating the GPCR CXCR4 are produced by standardtechniques. Lipoparticles are transiently permeabilized usingelectroporation. Electroporation causes pores to open in lipidmembranes, allowing entry of soluble, extracellular molecules. The poresreseal within milliseconds, leaving the particle intact. Lipoparticlesare suspended in a highly concentrated solution of purified Gα_(z) andelectroporated using five 1,000 V pulses of 10 msec duration each. Oneskilled in the art would recognize that alternative electroporationconditions may also be employed, including the use of between one andtwenty pulses of voltage between 100 and 2,000 V, each of 1 to 1,000msec duration. Unincorporated G protein is removed by passinglipoparticles through a sucrose cushion. The presence of G proteinswithin lipoparticles is verified by Western blot using anti-G proteinantibodies. One skilled in the art would recognize that additional oralternative G protein isotypes may be incorporated and that additionalor alternative transient or permanent permeabilization techniques may beemployed. Alternative permeabilization techniques include adding excessATP, EDTA, Ca++, Ca₃(PO₄)₂, DEAE-dextran, polyethylene-glycol, I-14402(a cell-loading reagent), S. aureus alpha-toxin, melittin, orstreptolysin-O. Alternatively, lipoparticles can be permeabilized byagitation, vortexing, or sonication.

Example 92 Incorporation of G Proteins into Lipoparticles Using GProtein Over-Expression

Lipoparticles incorporating the GPCR CXCR4 are produced by standardtechniques. During production the cells are cotransfected with a plasmidthat expresses the G protein Gz. Over-expression of Gz during GPCRincorporation into lipoparticles increases the likelihood of G proteinincorporation with the GPCR. The presence of G proteins withinlipoparticles is verified by Western blot using anti-G proteinantibodies. One skilled in the art would recognize that additional oralternative G protein isotypes could be incorporated.

Example 93 Incorporation of Fluorescent GPCR Fusion Proteins intoLipoparticles and Detection of Ligand-Mediated Activation of GPCRs

Cyan fluorescent-protein (CFP) is fused to the third intracellular loopof the transmembrane domain, and yellow fluorescent protein (YFP) fusedto the cytoplasmic tail of the GPCR CXCR4. Upon ligand binding, GPCRconformational changes bring the intracellular cytoplasmic tail of theGPCR within close proximity of the transmembrane domain loops, allowingfluorescence resonant energy transfer (FRET) from the donor proteinlabel to the acceptor, which subsequently emits a detectable signal, aspreviously demonstrated for other GPCRs (Vilardaga, et al. (2003), NatBiotechnol, 21:807-12). Fusion proteins are created using standardcloning methodology, and incorporated into lipoparticles using standardtechniques. The presence of the fusion protein within producer cells andlipoparticles is verified by Western blot using anti-CXCR4 antibodies. Gproteins and GTP are incorporated into the lipoparticles as describedherein.

To test its function, the labeled lipoparticles (approximately 1 νg) arestimulated with the CXCR4 agonist SDF-1 (60 nM). Ligand mediatedactivation of CXCR4 results in a conformational change in the GPCR,causing the emission of a detectable fluorescent signal from the CFP-YFPFRET pair. Co-treatment of lipoparticles with a CXCR4 antagonist, suchas the antibody 12G5, will inhibit this fluorescent signal.Lipoparticles lacking CXCR4 (null Lipoparticles) fail to exhibit anychange in fluorescence. Fluorescence is measured in real-time, beginningprior to addition of SDF-1, using a Perkin Elmer LS-50B fluorometer. A 2ml cuvette and 750 ul volume will be used, but other formats, such as amicroplate, could also be used. One skilled in the art would recognizethat additional or alternative GPCRs and fluorescent or luminescentproteins (e.g. FRET or BRET pairs) could also be fused and incorporatedinto lipoparticles in a similar manner.

Example 94 Detection of GPCR-G Protein Activation and Signaling byIncorporation of Fluorescently-Labelled GTP-γS into Lipoparticles

FL-GTP-γS is available in non-hydrolysable formats that bindirreversibly to G proteins, and in which the fluorescent signal isapproximately 90% quenched in the free, but not bound moiety. FL-GTP-γScontaining a variety of fluorophores are available (for example, theMANT or BODIPY fluorophores available from Molecular Probes). MANT isused in this experiment, but BODIPY, or other fluorescent orbioluminescent reporters could also be used. FL-GTP-γS will be loaded byelectroporation into pre-formed lipoparticles containing the GPCR CXCR4and a G protein, incorporated as described herein. lipoparticles aresuspended in a concentrated solution of purified FL-GTP-γS, andelectroporated using five 1,000 V pulses of 10 msec each. One skilled inthe art would recognize that alternative electroporation conditions mayalso be employed, and that alternative methods for incorporation ofFL-GTP-γS could also be used. These include, but are not limited to,transient poration using mild sonication, or permanent poration usingpeptides such as melittin. Unincorporated FL-GTP-γS is removed using aG50 spin column, and incorporated fluorescence is detected using aPerkin-Elmer LS-50B fluorometer. Controls will include lipoparticlesthat have not been electroporated.

To test their functional integrity, the labeled lipoparticles arestimulated with the CXCR4 agonist SDF-1. Stimulation of CXCR4 will causeGα to irreversibly bind FL-GTP-γS, allowing the MANT fluorophore to emita detectable fluorescent signal. Fluorescence emission is not altered innull-lipoparticles, or in lipoparticles treated with a CXCR4 antagonistsuch as the antibody 12G5. Fluorescence is measured in real-time,beginning prior to addition of SDF-1, using a Perkin Elmer LS-50Bfluorometer. One skilled in the art would recognize that alternativeguanine-nucleotides could be incorporated into lipoparticles in asimilar manner, including MANT-GTP, MANT-GMPPNP, BODIPY-FL-GTP,BODIPY-R6G-GTP, BODIPY-TR-GTP, BODIPY FL GMPPNP, BODIPY FL GTP-γ-Sthioester, TNP-GTP (2′-(or 3′-)O-(trinitrophenyl)guanosine5′-triphosphate), BzBzGTP (2′-(or 3′-)O-(4-benzoylbenzoyl)guanosine5′-triphosphate), S-(DMNPE-caged) GTP-γ-S, or Europium-GTPγ S. Oneskilled in the art would also recognize that the lipoparticles withinthis example could be modified prior to stimulation with agonist topermit signaling components to interact. Such modification couldinclude, but is not limited to, disruption by sonication, vortexing, ordetergent; or poration using melittin, Streptolysin-O, polyethyleneglycol, high amounts of calcium, or low amounts of alkanes.

Example 95 Incorporation of Fluorescently-Labeled G Proteins intoLipoparticles

YFP is fused to the carboxy-terminus of Gα_(z), and CFP fused to thecarboxy terminus of Gβγ. Fusion proteins are created using standardcloning methodology. Lipoparticles containing the GPCR CXCR4 areconstructed. The fusion proteins are incorporated into theselipoparticles as described herein. The presence of fusion proteins inproducer cells and lipoparticles is verified by Western blot usinganti-G protein antibodies. One skilled in the art would recognize thatadditional or alternative GPCRs, G proteins, and fluorescent proteinlabels could similarly be incorporated. To test their function, thelabeled lipoparticles are exposed to the CXCR4 agonist SDF-1. Ligandbinding by CXCR4 will stimulate dissociation of the heterotrimeric Gprotein complex, causing a reduction in the fluorescent emission fromthe CFP-YFP FRET pair. Fluorescently excited G-protein tryptophanresidues could act as an alternative donor for excitation of the FRETacceptor. Fluorescence emission is not altered in null-lipoparticles, orin lipoparticles treated with a CXCR4 antagonist such as the antibody12G5. Fluorescence is measured in real-time, beginning prior to theaddition of SDF-1, using a Perkin Elmer LS-50B fluorometer.

Example 96 Incorporation of Fluorescently-Labeled G Proteins and GPCRsinto Lipoparticles

YFP is fused to the carboxy-terminus of Gα_(z), and CFP fused to theC-terminus of CXCR4. Fusion proteins are created using standard cloningmethodology. Lipoparticles containing the CXCR4 fusion are constructedusing standard techniques. G protein fusions are incorporated into theselipoparticles using one, or a combination of the methods outlined inprevious Examples. The presence of fusion proteins in lipoparticles areverified by Western blot using anti-G protein and anti-GPCR antibodies.One skilled in the art would recognize that additional or alternativeGPCRs, G proteins, and fluorescent protein labels could similarly beincorporated. To test their function, the labeled lipoparticles areexposed to the CXCR4 agonist SDF-1. Ligand binding by CXCR4 stimulatesdissociation of the G protein from the GPCR, causing a reduction offluorescence from the CFP-YFP FRET pair. Fluorescence emission is notaltered in null-lipoparticles, or in lipoparticles treated with a CXCR4antagonist such as the antibody 12G5. Fluorescence is measured inreal-time, beginning prior to the addition of SDF-1, using a PerkinElmer LS-50B fluorometer.

Example 97 Incorporation of a Membrane Potential Sensor

Lipoparticles containing CXCR4, Gα_(z), and GTP are constructed.Lipoparticles are suspended in a concentrated solution of thefluorescent dye di-4-ANEPPS which diffuses preferentially into the lipidbilayer of cell membranes, and is incorporated into the Lipoparticles.Examples of alternative voltage-sensitive fluorescent dyes include, butare not limited to, di-4-ANEPPS(C₂₈H₃₆N₂O₃S), di-8-ANEPPS, rhodamine421, oxonol VI, JC-1, DiSC3(5), and the like (Molecular Probes, Inc.).The ANEPPS dyes can be measured ratiometrically, responding to increasesin membrane potential with a decrease in fluorescence excited atapproximately 440 nm and an increase in fluorescence excited at 530 nm.The presence of CXCR4 and Gα_(z) in lipoparticles is verified by Westernblot using anti-G protein and anti-GPCR antibodies. One skilled in theart would recognize that alternative GPCRs, G proteins, membranepotential-responsive fluorescent probes, or pH-responsive probes couldalso be used. To test their function, these lipoparticles are exposed tothe CXCR4 agonist SDF-1. Ligand binding by CXCR4 results in a change inthe structural conformation of the receptor associated with G-proteindissociation. This results in a change in the electrical potentialacross the lipoparticle membrane, causing a detectable signal emissionfrom the ANEPPS probe. Fluorescence emission is not altered innull-lipoparticles, or in lipoparticles treated with a CXCR4 antagonistsuch as the antibody 12G5. Fluorescence is measured in real-time,beginning prior to the addition of SDF-1, using a Perkin Elmer LS-50Bfluorometer.

Example 98 Incorporation of a Signaling Intermediate Ion Channel

Lipoparticles are constructed by standard techniques to contain CXCR4,Gα_(z), GTP, and the ion channel. The ion channel may be aninwardly-rectifying potassium channels (GIRK) such as Kir3.x. Thefluorescent dye di-4-ANEPPS is loaded into this lipoparticle aspreviously described. Di-4-ANEPPS fluoresces in a lipid environment andits fluorescence spectrum changes in response to fluctuations inmembrane potential. The presence of CXCR4 and Gα_(z) in lipoparticles isverified by Western blot using anti-G protein and anti-GPCR antibodies.One skilled in the art would recognize that alternative GPCRs, Gproteins, and membrane potential-responsive fluorescent probes couldalso be used. To test their function, the labeled lipoparticles areexposed to the CXCR4 agonist SDF-1. Stimulation of CXCR4 causesdissociation of the G protein from CXCR4 and activation of the ionchannel. The resultant movement of ions causes an alteration to thelipoparticle membrane potential, leading to a change in the fluorescenceof the di-4-ANEPPS. Fluorescence emission is not altered innull-lipoparticles or in lipoparticles treated with a CXCR4 antagonist,such as the antibody 12G5. Fluorescence is measured in real-time beforeand after adding SDF-1 using a Perkin Elmer LS-50B fluorometer.

Example 99 Comparison of Ligand Affinity for GPCR with and withoutIncorporated G Proteins

Radioligand binding curves are performed to detect ligand binding tolipoparticles and to estimate the affinity of their interaction. In somecases, the affinity of ligand binding is affected by whether the GPCR iscoupled or uncoupled from G proteins. The affinity change is a result ofthe GPCR exterior assuming different structural conformations in thepresence and absence of G proteins. To measure this phenomenon inlipoparticles, CXCR3 lipoparticles are prepared with and withoutincorporated G protein. Radiolabeled I-TAC (Perkin-Elmer) is used tobind lipoparticles containing the CXCR3 membrane protein. Lipoparticlesare resuspended in a total of 100 ul of Hepes⁺⁺ Binding Buffer (50 mMHepes 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 150 mM NaCl, and 0.5% BSA) togetherwith 0.1 nM radioligand. Increasing amounts of cold ligand (Peprotech)are also included. The mixture is incubated 1 h at room temperature andthen filtered through Whatman GF/C filters soaked in 0.2%polyethyleneimine (PEI). Filters are counted in a Wallac gamma counter.The results are plotted as a competition curve to determine the affinityof the ligand for the receptor in its coupled versus uncoupled state.

Example 100 Incorporation of an Antibody-Anchoring ZZ-TM FusionSensor/Reporter Protein into Lipoparticles

A 1-TM sensor/reporter is a fusion protein (ZZ-TM) comprising thefollowing contiguous domains (from amino- to carboxy-termini): twoStaphylococcal protein A Z-domains, the transmembrane domain of PDGFR,and a fluorescent protein (either the fluorescent donor CFP or thefluorescent acceptor YFP). When expressed within the membrane of alipoparticle, the exterior Z-domains of the ZZ-TM fusion are capable of‘capturing’ antibody by binding the constant (Fc) domains, andexpressing it in the correct orientation for target recognition andbinding. The internal fluorescent protein domains of the sensor/reporterwill undergo FRET if acceptor and donor pairs are brought into closeproximity by ZZ-TM clustering and cross-linking following targetbinding. One skilled in the art would recognize that the proteins couldalso be designed to undergo BRET.

The ZZ coding sequence is excised from pEZZ18 (codon optimized forexpression within human cells, Amersham), and ligated into the cloningsite of pDisplay (Invitrogen), a plasmid designed to facilitateexpression of soluble molecules on the cell surface by fusing them to aminimal PDGFR transmembrane domain, a leader sequence (for surfaceexpression), and epitope tags (for detection). The sequence formonomeric CFP or YFP is included at the 3′-end of the coding sequencefor ZZ-TM using standard cloning techniques. Lipoparticles containingpaired fusion proteins are produced in HEK-293 cells using methodspreviously described. Lipoparticles simultaneously contain bothfluorescent protein fusion proteins (ZZ-TM-CFP and ZZ-TM-YFP) in orderfor FRET to occur. The presence of the fusion protein in producer cellsand in lipoparticles is verified by Western blot using Fab fragmentprimary antibodies and anti-light chain secondary antibodies. Between 1and 100 of each ZZ-TM pair are incorporated into each lipoparticle.

One skilled in the art would recognize that additional methods ofincorporating functional membrane proteins into the lipoparticle arealso possible, including avidin-biotin linkage, amine coupling, geneticfusion of Fv fragments onto reporters, or incorporation of naturallyexpressed BCRs.

Example 101 Use of ZZ-TM Lipoparticles and a Monoclonal Antibody forPathogen Detection

Lipoparticles containing the ZZ-TM sensor/reporter fusion protein arecreated as described above. In order to make the ZZ-TM fusion proteinstarget-specific, a monoclonal antibody specific for DEN is ‘captured’ onthe ZZ-TM sensor/reporter protein following lipoparticle production.Solutions of anti-DEN E-protein antibody (containing from 1 pg to 1 μgof antibody) are added to aliquots of ZZ-TM lipoparticles (containing 1μg of protein) in Hepes buffered saline (HBS). After 30 minutes at 25°C., unbound antibody is removed by passing the lipoparticle suspensionsthrough sucrose cushions. One skilled in the art would recognize thatthe ZZ-TM sensor reporter is capable of binding polyclonal or monoclonalantibodies singly or in combination, and that the target-specificity ofthe ZZ-TM fusion can be modified by replacing the ‘captured’ antibodywith one with alternative epitope-recognition characteristics.Antibody-primed ZZ-TM lipoparticles are placed into stirred cuvettes in1 ml HBS, and from 1 pg to 1 μg (protein) of DEN monomeric E protein,dimeric E protein, or non-infectious virions expressing E protein isadded. Fluorescence is recorded in real-time using a Perkin-Elmer LS-50Bfluorometer (excitation 436 nm, ratiometric emission at 480 and 535 nm).Baseline fluorescence is determined using ZZ-TM lipoparticles withoutMAb, and using lipoparticles not expressing the ZZ-TM sensor/reporter.Heat-destroyed antigen, WNV E proteins, and ZZ-TM linked to irrelevantantibodies are used as negative controls. Anti-mouse secondaryantibodies are used as positive controls (will cross-link the capturedprimary antibodies). One skilled in the art would recognize that thebasic pathogen detection assay could be applied to any infectious agentby changing the specificity of the captured recognition antibody, andthat alternative detection formats or technologies (e.g. a microfluidicdevice, a flow cell, a Lab-on-a-Chip, a 96-well plate, a 384-well plate,a 1536-well plate, a glass slide, a plastic slide, an optical fiber, aflow cytometer, a microscope, a fluorometer, a spectrometer, or a CCDcamera) could also be used.

Example 102 Use of ZZ-TM Lipoparticles and Combination MonoclonalAntibodies for Pathogen Detection

An assay which utilizes multiple independent sensing and/or reportingmechanisms has a higher degree of fidelity and confidence than onerelying upon a single target-recognition element. The goal of thisexperiment is to use ZZ-TM lipoparticles bearing multiplepathogen-specific MAbs to detect the presence of pathogen in samples.The use of two MAbs able to recognize different epitopes on the sameantigen molecule increases the specificity of the pathogen detectionassay when compared with using a single MAb. Lipoparticles containingthe ZZ-TM sensor/reporter fusion protein are created as describedherein. ZZ-TM lipoparticles are made target specific by adding twomonoclonal antibodies with specificities to separate epitopes on thesame protein. The HIV Envelope protein gp120 from strain IIIB is used,and MAbs include D47 and b12, which recognize different(non-competitive) epitopes on the same protein (V3 loop and CD4-bindingsite, respectively). Briefly, solutions of each monoclonal antibody(from 1 pg to 1 μg) are simultaneously added to aliquots of ZZ-TMlipoparticles (containing 1 μg of protein) in HBS. Unbound antibody isremoved by passing lipoparticles through sucrose cushions. The basicpathogen detection assay described above is performed. Briefly,monomeric, oligomeric, or virion-expressed HIV Envelope protein is addedto aliquots of antibody-primed ZZ-TM lipoparticles in stirred cuvettesand fluorescence is measured in real-time. One skilled in the art wouldrecognize that the basic pathogen detection assay could be applied toany infectious agent or other antigen simply by changing the specificityof the captured recognition antibody. The MAbs may recognize epitopes onthe same protein or on different proteins, so long as simultaneousrecognition of both is sufficient to cross-link the ZZ-TM sensorprotein.

Example 103 Use of ZZ-TM Lipoparticles and Polyclonal Antibodies forPathogen Detection

The use of a polyclonal antibody increases the recognition diversity ofthe pathogen detection assay compared with using a single MAb.Lipoparticles containing the ZZ-TM sensor/reporter fusion protein arecreated as described above. ZZ-TM lipoparticles are made target specificby adding an anti-DEN polyclonal antibody, as described herein. Briefly,solutions of antibody (from 1 pg to 1 μg) are added to aliquots of ZZ-TMlipoparticles (containing 1 μg of protein) in HBS, and unbound antibodysubsequently removed by passing lipoparticles through sucrose cushions.The basic pathogen detection assay described above is performed.Briefly, monomeric, dimeric, or virion-expressed DEN E protein is addedto aliquots of antibody-primed ZZ-TM lipoparticles in stirred cuvettesand fluorescence is measured in real-time. One skilled in the art wouldrecognize that the basic pathogen detection assay could be applied toany infectious agent or other antigen simply by changing the specificityof the captured recognition antibody. Affinity chromatography could beused to concentrate antigen-specific subsets of the polyclonalpreparation.

Example 104 Use of ZZ-TM Lipoparticles and Antigen-IgG Fusions forAntibody Detection

Lipoparticles containing the ZZ-TM sensor/reporter fusion protein arecreated. ZZ-TM lipoparticles are made target specific by adding a DEN Eprotein-IgG fusion protein. This protein is created using standardcloning techniques by fusing the Fc portion of rabbit immunoglobulin Gto the receptor binding domain (domain III) of the DEN E protein(courtesy of Dr. Ted Pierson). Because it contains an Fc domain, theprotein can be bound by protein A (and the ZZ-TM sensor/reporter) in amanner similar to that for standard antibody molecules. Solutions of DENE-IgG (containing from 1 pg to 1 μg of protein) are added to aliquots ofZZ-TM lipoparticles (containing 1 μg of protein) in HBS. After 30minutes at 25° C., unbound E-IgG is removed by passing the lipoparticlesuspensions through sucrose cushions. Primed ZZ-TM lipoparticles areplaced into stirred cuvettes in 1 ml HBS, and from 1 pg to 1 μg(protein) of target antibody (anti-DEN E-protein) is added. Fluorescenceis recorded in real-time using a Perkin-Elmer LS-50B fluorometer(excitation 436 nm, ratiometric emission at 480 and 535 nm). Baselinefluorescence is determined using ZZ-TM lipoparticles without capturedE-IgG and/or using lipoparticles not expressing the ZZ-TMsensor/reporter. Heat-destroyed antibody, irrelevant IgG-proteinfusions, and/or irrelevant antibodies in place of the target antibodyare used as negative controls. One skilled in the art would recognizethat the basic antibody detection assay could be applied to any targetantibody simply by changing the epitope nature of the capturedantigen-IgG.

Example 105 Incorporation of an Antibody Fab Fragment-Anchoring B1-TMFusion Sensor/Reporter Protein into Lipoparticles

The goal of this experiment is to incorporate a 1-TM protein intolipoparticles, capable of target recognition (e.g. pathogen, antibody,or ligand binding), of clustering and cross-linking upon target binding,and of reporting target binding/cross-linking (e.g. by participation inFRET). The 1-TM sensor/reporter is a fusion protein (B1-TM) comprisingthe following contiguous domains (from amino- to carboxy-termini): animmunoglobulin light-chain-binding domain (B1) of Peptostreptococcusmagnus Protein L, the transmembrane domain of PDGFR, and a fluorescentprotein (CFP or YFP). When expressed in lipoparticles, the externalB1-domain is capable of ‘capturing’ target-specific Fab fragments, andpresenting them in the correct orientation for target recognition andbinding. The fluorescent protein domains undergo FRET if acceptor anddonor pairs are brought into close proximity following B1-TM clusteringand cross-linking upon antigen binding. One skilled in the art wouldrecognize that alternative 1-TM backbones (e.g. CD4, Tva, EGFR), andalternative fluorescent proteins could also be used to create the B1-TMsensor/reporter. The B1-TM sensor/reporter fusion protein andlipoparticles containing it are created using standard molecular cloningtechniques, as described for the production of the ZZ-TM lipoparticles.The B1-TM protein provides a sensor with a different target-bindingstoichiometry than the ZZ-TM protein because B1 binds monomeric Fabfragments, whereas each Z domain binds dimeric antibodies. The presenceof the fusion protein in producer cells and in lipoparticles is verifiedby Western blot.

Example 106 Use of B1-TM Lipoparticles and Fab Fragments for PathogenDetection

The goal of this experiment is to use B1-TM lipoparticles bearing Fabfragments to detect the presence of pathogen in samples. Lipoparticlescontaining the B1-TM sensor/reporter fusion protein will be created asdescribed above. B1-TM lipoparticles are made target specific by addingan anti-DEN Fab fragment in a manner similar to that described above.Briefly, Fab solutions (from 0.25 pg to 0.25 μg) are added to aliquotsof B1-TM lipoparticles (containing 1 μg of protein) in HBS, and unboundFab fragments are subsequently removed by passing lipoparticles throughsucrose cushions. The basic pathogen detection assay described above isperformed. Briefly, monomeric, dimeric, or virion-expressed DEN Eprotein is added to aliquots of Fab-primed B1-TM lipoparticles instirred cuvettes and fluorescence measured in real-time. One skilled inthe art would recognize that any Fab fragment could be incorporated intothe sensor/reporter, and that the basic pathogen detection assay couldbe applied to any infectious agent or other antigen simply by changingthe specificity of the captured recognition Fab fragment.

Example 107 Incorporation of a 1-TM Fusion Sensor/Reporter Protein intoLipoparticles, and its Use to Detect Receptor Ligands

The goal of this experiment is to incorporate into lipoparticles a 1-TMreceptor capable of target recognition, ligand-dependent clustering, andreporting of ligand binding. These lipoparticles are used for assaydetection of ligands in samples. The 1-TM receptor comprises theepidermal growth factor receptor (EGFR) fused at its intracytoplasmicdomain to a fluorescent protein (YFP or CFP). EGFR exists as a monomerwhen unliganded, but clusters and cross-links following binding by itscognate ligand, EGF. A plasmid containing the entire coding sequence forEGFR fused at its carboxy-terminus to YFP or CFP is created by standardcloning techniques. The fluorescent protein domains undergo FRET whenboth are incorporated into the same lipoparticle and acceptor and donorpairs are brought into close proximity by 1-TM receptor clustering andcross-linking following ligand binding. One skilled in the art wouldrecognize that alternative 1-TM receptors, or alternative fluorescent orluminescent protein domains could also be incorporated intolipoparticles. Lipoparticles containing the 1-TM fusion sensor/reporterare produced by standard procedures as described herein. The presence ofthe fusion protein in producer cells and in lipoparticles is verified byWestern blot.

For ligand detection, aliquots of 1-TM lipoparticles (containing 1 μg ofprotein) are placed into stirred cuvettes, and 1 pg to 1 μg of targetligand (dimeric EGF) in 1 ml HBS is added. Fluorescence is recorded inreal-time using a Perkin-Elmer LS-50B fluorometer (excitation 436 nm,ratiometric emission at 480 and 535 nm). Baseline fluorescence isdetermined using lipoparticles not expressing the 1-TM sensor/reporter.Heat-destroyed ligand, and/or irrelevant ligands are used as negativecontrols. Anti-EGFR antibodies replacing the target ligands are used aspositive controls. One skilled in the art would recognize that thisligand detection assay could be applied to any ligand simply by varyingthe receptor component of the 1-TM sensor/reporter fusion protein.

Example 108 Use of Lipoparticles Containing ZZ-TM and 1-TMSensor/Reporters to Detect Ligand

The goal of this experiment is to use lipoparticles containing both aZZ-TM and a 1-TM sensor/reporter to detect the presence of a targetligand in samples. The use of antibody-bearing ZZ-TM sensor/reporters incombination with 1-TM sensor/reporters will increase the specificity ofthe ligand detection assay compared with using one sensor/reporteralone. ZZ-TM antibody-capture fusion proteins containing a YFP domainare constructed. A 1-TM receptor (EGFR) fused to CFP is constructed asdescribed above. One skilled in the art would recognize that alternative1-TM domains, or alternative fluorescent or luminescent protein domainscould also be incorporated into lipoparticles. Lipoparticles containingboth of these sensor/reporters are produced by standard methods asdescribed herein. ZZ-TM sensor/reporters are made target specific by theaddition of an anti-EGF MAb. The fluorescent protein domains undergoFRET when acceptor and donor pairs are brought into close proximity byMAb-ZZ-TM and 1-TM receptor binding to the same ligand molecule.Aliquots of the antibody-primed ZZ-TM/1-TM lipoparticles (containing 1μg of protein) are placed into stirred cuvettes, and 1 pg to 1 μg of EGFin 1 ml HBS added. Fluorescence is recorded in real-time using aPerkin-Elmer LS-50B fluorometer (excitation 436 nm, ratiometric emissionat 480 and 535 nm). Baseline fluorescence is determined usinglipoparticles not expressing the sensor/reporter proteins.Heat-destroyed ligand and/or irrelevant ligands are used as negativecontrols. One skilled in the art would recognize that the basic liganddetection assay could be applied to any ligand simply by varying thereceptor component of the 1-TM sensor/reporter and the antibodyspecificity of the ZZ-TM sensor/reporter, and that alternative detectionformats (e.g. flow cytometry, optical biosensors) could also be used.The fluorescent protein CFP can be fused to either the 1-TM protein oron the ZZ-TM protein, so long as an appropriate acceptor protein (YFP)is fused to the complementary protein.

Example 109 Detection of Pathogens Using Flow-Cell Based Technology

Lipoparticles containing the ZZ-TM fusion protein are produced asdescribed above. The ZZ-TM sensor/reporters are made target specific bycapturing an anti-DEN E protein MAb as described above. Monomeric ordimeric DEN E protein, or virions expressing DEN E protein areamine-coupled to a carboxymethyldextran surface. Alternative couplingtechniques, such as, but not limited to capture with a specific antibodyor avidin-biotin interactions could also be used and other biochipsurfaces could also be used. A suspension of antibody-primed ZZ-TMlipoparticles (1 pg to 1 μg) in HBS is passed through a flow-cell andfluorescence is monitored in real-time using ultra-sensitive assaysbased on evanescent field excitation (commonly known as TIRF) (Evans, etal. (2003), Neuron, 38:145-7, Wakelin, et al. (2003), J Microsc,209:143-8). Fused silicon microscope slides derivatized withcarboxymethyldextran are mounted on an inverted microscope stage in asmall-volume (100 μl) lab-assembled flow cell that allows control overthe injection and flow of analyte solutions. Derivitization commonlyinvolves aminopropylsilanization followed by an EDC/NHS reaction ofcarboxymethyldextran with this aminated surface (Wakelin, et al. (2003),J Microsc, 209:143-8). Fluorescence excitation of captured lipoparticlesensors is induced by the evanescent field of a prism-coupled,internally reflected light source. Fluorescence emission is collectedvia a microscope objective focused on the derivatized slide surface anddetected by a microscope mounted camera through appropriate emissionfilters. Alternatively, one skilled in the art would recognize a similarapproach through the employment of derivatized fiber optics forevanescent excitation and collection of fluorescence with a sheath-basedflow cell (Epstein, et al. (2003), Chem Soc Rev, 32:203-14, Marazuela,et al. (2002), Anal Bioanal Chem, 372:664-82). Negative controls includeflow cells to which no antigen has been coupled and/or lipoparticlescontaining no ZZ-TM sensor/reporter. Variations based on this basicpathogen-detection assay are possible, such as coupling of thelipoparticles to the biochip and flowing solutions of the antigenthrough the flow cell. The assay could be applicable to any infectiousagent or other antigen, simply by varying the specificity of theantibody captured by the ZZ-TM sensor/reporter.

Example 110 Detection of Target Protein in Tissue Sections

Lipoparticles containing the ZZ-TM fusion protein are produced asdescribed above. The ZZ-TM sensor/reporters are made target specific bycapturing an anti-mouse megalin MAb as described above. Megalin is amembrane protein found in a variety of tissues, including renal tubules,intestine, and thyroid, where it facilitates the trans-epithelialtransport of a variety of hormones, vitamins and other molecules. Fixedsections of mouse kidney are incubated in suspensions of antibody-primedZZ-TM lipoparticles (containing from 1 pg to 1 μg protein) at 37° C. for1 hour. Sections are washed and visualized by fluorescent microscopy todetect FRET-induced signals. This basic immunohistochemistry procedurecould be applied to any cell target simply by varying the specificity ofthe antibody captured by the ZZ-TM sensor, or by using a target-specific1-TM sensor/reporter.

Example 111 Detection of Lipoparticle Signaling by ProteinReconstitution

This example detects 1-TM (single transmembrane) receptor signaling inlipoparticles using protein fragment complementation. The basis forprotein fragment complementation assay is the reconstitution of afunctional protein, such as an enzyme or a fluorescent protein, fromrationally-designed inactive fragments fused to target proteins, such asmembrane receptors. Interaction of the target proteins allows thefolding of the reporter fragments into a functional protein, theactivity of which is then detected. Two fusion proteins are constructedusing standard cloning techniques. One fusion protein consists of theextracellular and transmembrane domains of the 1-TM receptor EGFR, withone half of the fluorescent protein GFP fused at its cytoplasmic tail(EGFR/GFPf1). The other fusion protein consists of identical EGFRdomains fused to the complementary half of GFP (EGFR/GFPf2).Lipoparticles, simultaneously expressing both of these fusion proteins,are produced by methods already described, and suspended in HBS. Anantibody recognizing EGFR will be added, and fluorescence measured in astirred cuvette fluorometer. One skilled in the art would recognize thatalternative transmembrane domains, including PDGFR, EPOR, and Tva couldalso be used, and that other inactive but complementary protein reporterfragments, including alternative fluorescent proteins (e.g. CFP, YFP),and enzymes (luciferase, β-lactamase, dihydrofolate reductase) couldalso be included (Galarneau, et al. (2002), Nat Biotechnol, 20:619-22,Luker, et al. (2004), Methods Enzymol, 385:349-60, Massoud, et al.(2004), Faseb J, Michnick (2001), Curr Opin Struct Biol, 11:472-7,Pelletier, et al. (1999), Nat Biotechnol, 17:683-90, Pelletier, et al.(1998), Proc Natl Acad Sci USA, 95:12141-6, Remy, et al. (2004), MolCell Biol, 24:1493-504).

Example 112 Incorporation of Gag/Fluorescent Protein Fusion Reportersinto Lipoparticles

Gag is the one retroviral protein necessary and sufficient forlipoparticle production. In normal retroviruses, Gag is normallyexpressed as a fusion protein with enzymatic proteins (Pol). However, asPol proteins are not necessary for lipoparticle formation, theirsequence can be substituted with a variety of alternative genes,including those for fluorescent reporter proteins (Bennett, et al.(1991), J Virol, 65:272-80, McDonald, et al. (2002), J. Cell Biol., 159:McDonald, et al. (2003), Science, 300:1295-7, Weldon, et al. (1990), JVirol, 64:4169-79). Approximately 1,000-2,000 Gag proteins form thestructural core of each lipoparticle (Knipe, et al. (2001)), so proteinsfused to Gag are highly represented.

Standard cloning techniques were used to fuse enhanced GFP (eGFP) to theC-terminus (base pair 1955) of the MoMLV Gag protein. Lipoparticlescontaining the Gag/GFP fusion were produced in HEK293 cells usingmethods described herein. The Gag/GFP fusion protein was evaluated bymolecular weight confirmation using Western blot analysis, and itssuccessful incorporation into lipoparticles was confirmed by directmicroscopic visualization of lipoparticle fluorescence (FIG. 20) usingepifluorescent illumination under an oil-immersion 100× lens (Nikon).Similarly, Gag/CFP and Gag/YFP fusion proteins were also created andincorporated into lipoparticles (FIG. 20). Lipoparticles were alsoconstructed with both Gag/CFP and Gag/YFP simultaneously byco-expressing the Gag fusion proteins during production. Incorporationof both fluorophores was verified by fluorescence of both CFP and YFP inthese particles as measured with an LS-50B Perkin Elmer fluorometer.

One skilled in the art would recognize that additional fluorescent (e.g.Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), BlueFluorescent Protein (BFP), Cyan Fluorescent Protein (CFP), DsRED, AsRED,AmCyan, HcRed, ZsGreen, ZsYellow, or variants thereof) andnon-fluorescent proteins (e.g. esterases, proteases, kinases, Gproteins, alkaline phosphatase, peroxidase, beta lactamase) could besimilarly incorporated into lipoparticles by fusion to Gag, and thatfluorescent and non-fluorescent proteins could be fused to otherlipoparticle-integrated proteins (either native viral or artificiallyexpressed proteins), and thus be co-incorporated, either individually orin combinations.

Example 113 Incorporation of Membrane Protein/Fluorescent Protein FusionReporters into Lipoparticles

GFP was fused to the carboxy-(cytoplasmic) terminus of CXCR4 usingstandard cloning strategies. Lipoparticles containing the CXCR4/GFPfusion protein were produced in HEK293 cells using methods describedherein. The presence of the CXCR4/GFP fusion within producer cells andlipoparticles was verified by direct microscopic visualization offluorescent lipoparticles (FIG. 20). Three other membrane proteins fusedto GFP were similarly incorporated into lipoparticles, including CCR5,CXCR3, and cannibanoid receptor 1 (CB1) (FIG. 20).

One skilled in the art would recognize that additional fluorescent andnon-fluorescent proteins, including alternative fluorescent proteins(e.g. CFP, YFP, dsRed) and enzymes (e.g. alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and other oxidases, kinases,and proteases), could be incorporated into lipoparticles by similarlyfusing them to CXCR4 or other membrane proteins (e.g. CCR5, CD4).

Example 114 Incorporation of Lipophilic Dyes into Lipoparticle Membranes

Lipophilic dyes preferentially partition into lipid substrates, such asphospholipid membranes, simplifying their incorporation intolipoparticles when compared to water-soluble dyes. The membranepotential probe di-4-ANEPPS (Montana, et al. (1989), Biochemistry,28:4536-4539, Rohr, et al. (1994), Biophysical Journal, 67:1301-1315,Venema, et al. (1993), Biochim Biophys Acta, 1146:87-96) was obtained asa powder, dissolved in ethanol, and added to an aqueous suspension oflipoparticles to a final concentration of 5 μM. Incorporation of the dyeinto lipoparticle membranes, which occurs almost instantaneously, wasevaluated by fluorescent microscopy. Four other lipophilic dyes weresimilarly incorporated into lipoparticles, including octadecylrhodamine, phosphatidyl choline-NDB, Nile Red, and diI (FIG. 20). Incases where the dye demonstrated high background, excess dye was removedby purification of the lipoparticles through a G50 spin column.

One skilled in the art would recognize that alternative strategies forthe incorporation of di-4-ANEPPS and numerous other lipophilic dyes orother lipophilic substances (e.g. labeled or unlabeled fatty acids,phospholipids, sphingolipids, steroids, triacylglycerols, octadecylrhodamine, lipophilic fluoresceins, coumarins, dialkylcarbocyanineprobes, and dialkylaminostyryl probes) could also be employed to obtainsimilar results.

Example 115 Incorporation of AM-Ester Dye Reporters into LipoparticleInteriors

The lipid bilayers of cell membranes (and lipoparticles) are normallyimpermeable to hydrophilic molecules such as water soluble fluorescentdyes. However, by coupling them to acetoxymethyl ester (AM-ester)groups, water soluble dyes can freely diffuse through lipid bilayers.This approach is commonly used to introduce water-soluble fluorescentdyes into cells, where they become trapped after removal of the AM-estergroup by the action of cellular esterases. The same strategy is employedto load water-soluble dyes into modified, esterase-containinglipoparticles. A Gag/esterase fusion protein is generated by standardcloning techniques, using a human hepatic carboxylesterase (Pindel, etal. (1997), J Biol Chem, 272:14769-75). Gag/esterase-lipoparticles areproduced in HEK293 cells using methods already described. The presenceof the Gag/esterase fusion within lipoparticles is verified by Westernblot analysis for the Gag fusion protein. Gag/esterase-lipoparticles aresuspended in an aqueous solution of 10 μM calcein-AM (Molecular Probes).Fluorescence of calcein is quenched in its AM-ester form, but isreleased when the ester bond is cleaved. The removal of ester groupsfrom calcein-AM molecules as they diffuse into lipoparticles ismonitored by measuring increased fluorescent emission in astirred-cuvette fluorometer. Retention of calcein within lipoparticlesis confirmed by direct visualization using fluorescent microscopy.Lipoparticles not containing an esterase serve as negative controls.

One skilled in the art would recognize that alternative esterases couldbe incorporated into lipoparticles using this or similar techniques, orby fusion to other lipoparticle protein constituents. One skilled in theart would recognize that alternative water-soluble fluorescent AM-esterdyes (e.g. Fura-2, SNARF-1, SBFI, PBFI, DAF-FM, or other AM-ester dyeslisted in the Molecular Probes handbook, all of which are incorporatedby reference herein (Haugland (2003))) could be incorporated intoGag-esterase lipoparticles individually or in combinations by similarmeans.

Example 116 Mechanical Encapsulation of Membrane-Impermeable Reportersinto Lipoparticles

Non-lipophilic probes and reporters that cannot be chemically modifiedto enable diffusion across lipid bilayers require mechanical deliveryinto membrane-bound spaces. Quantum dots (lipid membrane-impermeable,fluorescent reporters) are 3-6 nm spheres exhibiting high intensity andlong-lived fluorescent characteristics. Quantum dots are incorporatedinto lipoparticles using electroporation, which causes pores(approximately 10 nm) to transiently open (for millisecond periods) inlipid membranes, allowing entry of external molecules and particles.lipoparticles are suspended in solutions of concentrated quantum dotsand subjected to electroporation (200-2,000 V, 1-5 pulses of 1-20 msecduration each), conditions previously used for cells and liposomes(non-living, lipid spheres). Excess, non-incorporated quantum dots areremoved by passing lipoparticles through sucrose cushions, andlipoparticle-incorporated fluorescence is visualized by microscopy andquantified using a fluorometer. Controls can include lipoparticles thathave not been electroporated. One skilled in the art would recognizethat alternative membrane-impermeable fluorescent and non-fluorescentmolecules and particles (e.g. Phen-Green, lucigenin, OPA, radioactivity,paramagnetic beads, Raman probes (DSNB), gold) could be encapsulated inlipoparticles using similar strategies.

Example 117 Incorporation of Modified Lipids into Lipoparticle Membranes

Phospholipids, the major structural constituent of cell membranes, areglycerol-derived molecules comprised of a phosphorylalcohol molecule(the polar head), and two fatty acyl groups (non-polar tails). Whenphospholipids in solution are exposed to lipid membranes (such as cellmembranes, liposomes, and lipoparticles), their non-polar tailspartition into the membrane, leaving the polar head group, and moleculesconjugated to it, exposed on the membrane surface. A variety ofmolecules, including proteins such as avidin, and small molecules suchas biotin, may be conjugated to the phosphorylalcohol group ofphospholipids.

Lipoparticle surfaces were biotinylated by allowing biotin-conjugatedphosphoethanolamine (biotin-PE, Avanti Polar Lipids) to partition intolipoparticle membranes. To determine optimal lipoparticle biotinylationconditions, aliquots of lipoparticles were mixed with increasing amountsof biotin-PE at molar ratios of 1 thousand to 2.5 million (relative tolipoparticles), and reactions allowed to equilibrate for 1 hour. Thefunctional characteristics of biotin-PE lipoparticles were thenassessed.

The ability of biotinylated lipoparticles to bind avidin derivatives insolution was evaluated. Lipoparticles were immobilized on glass slidesby adsorption, excess biotin-PE removed by washing, and non-specificadsorption sites blocked for 20 minutes using 0.1% solutions of BSA. Theslides were then exposed to a molar excess of fluorescently labeledstreptavidin (50 nM in HBS) for 30 minutes and washed several times withbuffer. Lipoparticles were imaged at 100× magnification on a microscope,and their luminosity determined from digital images using Canvas (Denebasoftware) image analysis software. Curves describing the dynamics oflipoparticle incorporation of biotin-PE were constructed by plottingaverage lipoparticle luminosity (directly proportional to the molarconcentration of incorporated biotin-PE), against the total molarconcentration of biotin-PE used in each biotinylation reaction. Theincorporation of biotin-PE by fixed numbers of lipoparticles was foundto be both concentration dependent and saturable (FIG. 21).

The ability of biotinylated lipoparticles in suspension to be capturedby avidin-coated substrates was then evaluated. High-capacityprotein-binding wells were coated with NeutrAvidin (Pierce), adeglycosolated version of avidin with a near-neutral pI to minimizenon-specific binding, or BSA overnight. Lipoparticle/biotin-PE solutions(as above) were placed in the wells, which were centrifuged (1000 g, 90min) before unbound particles were removed by washing. Binding ofLipoparticles by the neutravidin wells was quantified using an assay forthe viral core protein, Gag. Neutravidin-bound lipoparticles werereleased and lysed using Triton X-100, transferred to new high bindingELISA plate wells, and allowed to adsorb to the well surfaces overnight.Total Gag concentration (proportional to the original neutravidin-boundlipoparticle concentration) was determined by ELISA using a rabbitanti-Gag primary antibody, an HRP conjugated anti-rabbit IgG secondaryantibody, and Pico chemiluminescent substrate (Pierce). Theconcentration of bound lipoparticles was then plotted against thelipoparticle:biotin-PE ratio used in each biotinylation reaction (FIG.21). Binding of biotinylated lipoparticles was biotin-PEconcentration-dependent to a maximum (a molar ratio of approximately1:100 000) after which binding decreased, presumably due to competitiveinterference by excess, unincorporated biotin-PE.

This demonstrates that a molar ratio of lipoparticle:biotin-PE between1:20,000 and 1:100 000 produced optimal avidin binding by biotinylatedLipoparticles. One skilled in the art would recognize that alternativebiotin-conjugated lipids or amphiphiles, differing, among other ways, inthe structure of the lipid-biotin linkages, lipid head-group, acyl chainlength, number, or structure, or non-glycerol based lipids, could alsobe used to biotinylate lipoparticle surfaces in a similar manner, andthat other molecules such as enzymes, small molecules, or fluorescentproteins, when coupled to phospholipids may also be linked tolipoparticle membranes in a similar manner.

Example 118 Covalent Attachment of Molecules to Lipoparticle Surfaces

Cell membranes, including those from which lipoparticles are derived,naturally contain protein and carbohydrate constituents that can benon-specifically targeted by reactive molecules that form covalent bondswith molecular groups such as aldehydes and amines. Lipoparticlesurfaces were biotinylated in this way using amine coupling. Aliquots oflipoparticles were suspended in buffered solutions (PBS pH 8.0 buffer)containing approximately 1,000-fold excess (relative to the totallipoparticle protein content)sulfo-NHS-biotin (amine reactive). After 1hour, excess biotin was removed using size exclusion (Sephadex G50) spinchromatography, using a buffer containing 0.1% Pluronics F127 to reducenon-specific binding of the lipoparticles. Biotinylation oflipoparticles was evident by specific binding of fluorescently labeledstreptavidin, which recognized lipoparticles only when biotinylated(FIG. 22). Lipoparticles were adsorbed to a microscope coverslip,blocked with 3% BSA, and exposed to a 50 nM solution ofAlexa-Streptavidin (Molecular Probes) in a 100 μl volume for 20 minutesbefore the surface was rinsed three times with 10 mM Hepes pH 7 100 mMNaCl and imaged. Both chemically and lipid (DPPE-biotin) biotinylatedlipoparticles produced punctate fluorescent images throughAlexa-Streptavidin binding, whereas non-biotinylated lipoparticles didnot bind the fluorescent protein. The same coverslip surface exposed tolipid biotin alone did not demonstrate any streptavidin binding.

Lipoparticles were similarly biotinylated using a carbohydrate-reactivebiotin kit (Pierce) that links biotin to surface carbohydrates on thelipoparticle by biotin-hydrazide (carbohydrate reactive, pH 5.5 MESbuffer). Lipoparticles were also biotinylated by mixing lipoparticleswith biotinylated WGA (Vector Laboratories). The WGA lectin bindsstrongly to the lipoparticle surface. Biotinylated-WGA binding tolipoparticles was evident through enhanced lipoparticle attachment tostreptavidin coated BIACORE chips during biosensing experiments.

Other reactive groups or methods for linking biotin to the Lipoparticlesurface could also be used, including, but not limited to, carboxyl orsulfhydryl-coupling to membrane proteins, or the use ofbiotin-conjugated proteins with an affinity for Lipoparticle surfacemoieties, such as biotin-conjugated lectins. One skilled in the artwould recognize that minor alterations to buffer composition, reactantratios, reaction conditions and reaction duration could result incomparable products. In addition to biotin, other molecules, includingmagnetic particles, quantum dots, gold particles, or radioactivesubstances, could also be linked to lipoparticle surfaces using similarapproaches. These other substances could be used to providelipoparticles with unique interactive and detection properties andfunctions, such as detection by MRI, detection by electron microscopy,or detection by radiography or PET.

Example 119 Linkage of Reporters to the Lipoparticle Exterior

Streptavidin-coated quantum dots (Quantum Dot Corp) are added toaliquots of biotinylated lipoparticles, and allowed to bind to thelipoparticle surface. Unbound quantum dots are removed by passinglipoparticles through a sucrose cushion. Fluorescence of thelipoparticles is visualized by microscopy and quantified using afluorometer. Controls can include non-biotinylated Lipoparticles. Oneskilled in the art would recognize that other avidin-, streptavidin-, orneutravidin-bound molecules or reporters could be linked to the surfaceof biotinylated lipoparticles in a similar manner. One skilled in theart would recognize that a lipoparticle expressing avidin orstreptavidin on its surface could be used similarly to ‘capture’biotinylated targets. One could also label with fluorescent lectin WGAor biotinylated lectin WGA. Other materials and proteins could also belinked to the lipoparticle exterior, such as antibodies.

Example 110 Incorporation of Enzymes into Lipoparticles

Enzymatic reporters, such as luciferase, catalyze reactions resulting inthe detectable chemical modification of a substrate. Luciferase is oneof the most sensitive reporters known (detectable to 10⁻²⁰ moles underideal conditions). Luciferase-containing lipoparticles were created byproducing lipoparticles using a Gag/luciferase fusion protein asdescribed herein for Gag-GFP fusion protein lipoparticles. In order totest the functional activity of the incorporated enzyme, lipoparticleswere permeabilized using small amounts of detergent (1% Triton X-100) toallow access of substrate and co-factors (luciferin, ATP, coenzyme A) toluciferase. Permeabilized lipoparticles were suspended in a substratebuffer (Promega Steady-Glo Luciferase Assay System), and luciferaseactivity was measured using a Wallac Victor2V luminometer. Permeabilizedlipoparticles not containing luciferase, and lipoparticles suspended inbuffer solutions without the substrate, were used as negative controls.Results using increasing amounts of lipoparticles demonstrated thatluciferase-containing lipoparticles retained luciferase activity, andthat lipoparticles without luciferase did not contain any substantialluciferase activity (FIG. 23). Lipoparticles were also tested withoutpermeabilization and did not display any significant luciferaseactivity. Lipoparticles were also permeabilized using melittin (apore-forming peptide), freeze-thawing, Lubrol, NP-20, CHAPS, andbeta-octylglucoside.

One skilled in the art would recognize that alternative means ofphysically associating enzymes with lipoparticles, such as fusion toother protein constituents including membrane proteins, as outlinedherein, incorporation of enzymatic membrane proteins such as BACE, orlinkage to the lipoparticle surface such as via biotin-streptavidininteractions as outlined herein, could also be used. Internallyencapsulated enzymes could also be cleaved from Gag, or other proteinsto which they are fused, if a protease target site is incorporated intothe fusion linker, and a protease such as MoMLV Pol is also included inthe lipoparticle. One skilled in the art would recognize thatalternative signaling, or non-signaling enzymatic proteins (e.g.alkaline phosphatase, GTPases, ATPases, horseradish peroxidase,beta-galactosidase, DNA and RNA polymerases, lipid-active enzymes suchas phospholipase A and C, and other oxidases, kinases, and proteases)could be incorporated in a similar manner, either individually or incombinations. One skilled in the art would recognize that alternativemethods for poration of lipoparticles could also be employed, includingsonication, vortex mixing, use of pore-forming peptides (e.g. melittin,streptolysin-O), polyethylene glycol, and low concentrations of alkanes.

Example 111 Poration and Permeabilization of Lipoparticles

Lipoparticles containing appropriately sized pores would be permeable tosmall molecules that would not normally be capable of crossing the lipidbilayer, while larger molecules, such as Gag or trapped reporters, wouldremain caged within the Lipoparticle. Melittin is a cationic,amphipathic peptide derived from the venom of the European honeybee. Itpartitions to the aqueous-lipid interface of cell membranes where itinduces disruption of the lipid bilayer, aggregation (pore formation),and formation of voltage-gated channels. It has been used in largeunilamellar vesicles (100 nm) to create pores with estimated radii ofbetween 0.3 to 5 nm (Rex, S. 1996. Biophys Chem 58: 75-85).

Lipoparticles containing a Gag-luciferase fusion protein were preparedas described herein. Melittin, ranging from 0.3 to 105 uM in finalconcentration, was added to solutions of Gag/luciferase-lipoparticles(MOPS pH 6.5 buffer), mixed, and incubated for 30 minutes. A 5 μlaliquot of the Lipoparticle solution was then transferred to a 96-wellplate containing 50 μl of Luciferase assay reagent (Promega Steady GloLuciferase Assay System) which contains the necessary reagents forenzymatic activity, including luciferin, ATP, and Mg²⁺. Luminescence wasmeasured for 1 second integrations in a Wallac Victor2V. When sufficientmelittin was added to the Lipoparticles, pore formation resulted ininflux of luciferin and ATP into the lipoparticle interior, and anincrease of the luminescent signal (FIG. 24). Triton X-100 was used tolyse luciferase-lipoparticles as a positive control.

One skilled in the art would recognize that numerous means oftransiently or permanently permeabilizing or porating lipoparticlescould be employed to similar effect. These include, but are not limitedto sonication or vortexing, laser-generated stress waves (Graier W F, etal. 1998. J Physiol 506, 109-125), use of pore-forming proteins andpeptides (e.g. streptolysin-O, aerolysin, maltoporin), pore-formingchannels (P2X7), polyethylene glycol, concentrated calcium, and lowconcentrations of alkanes. The pore-forming channel P2X7 could also beincorporated into lipoparticles. P2X7 creates a 900 Da pore uponaddition of 5 mM ATP (or BzBzATP or BzBzGTP) and can then re-close whenexposed to divalent cations such as magnesium or calcium. Permeabilizedlipoparticles could act as targeted or untargeted probes of localmicroenvironments, by allowing the interaction of trapped reporters withsmall molecules in the local vicinity.

Example 112 Linkage of Lipoparticles to Beads

Wheat Germ Agglutinin (WGA) is a dimeric, 36 kDa protein whichselectively binds the terminal N-acetylglucosamine of oligosaccharidesincluding glycolipids that occur abundantly in most mammalian cellmembranes (and lipoparticles). WGA, bound to 4% cross-linked agarosebeads are available from a variety of vendors, including VectorLaboratories. In order to demonstrate the ability to link lipoparticlesto these beads, 10 μl of fluorescent GFP-LipoProbes (lipoparticlesconstructed using a GFP/Gag fusion protein) were diluted to 100 μl inHBS buffer and mixed with 3 μl of either WGA agarose beads, or controlProtein A (ProA) agarose beads and allowed to incubate with gentlerocking for 30 minutes. The beads were spun for 2 minutes at 3 k rpm,and washed with 1 ml of HBS buffer. Binding of LipoProbes to the beadswas monitored directly by fluorescent microscopy (FIG. 25). LipoProbeswere observed to bind the WGA agarose beads, but not the control beads.

One skilled in the art would recognize that a variety of core beadmatrices could be used as alternatives to 4% agarose, including otheragarose formats, polymers such as polystyrene, divinylbenzene, andpolyvinyltoluene, poly-Lysine, and latexes such as sulfate-, carboxyl-,and chloromethyl-latex. Alternative bead-surface coupling groups to WGAcould also be used, including, but not limited to other lectins such asConA and DBA, aldehyde- and amine-reactive groups, ATP binding sites,carboxyl- and hydroxyl-reactive groups, thiols, phenyls and imidazoles,antibodies and other binding proteins, Protein A, Protein G, Protein L,and streptavidin. Specialty core matrices possessing intrinsic couplingand reporting properties, such as dyed or fluorescent microspheres,quantum dot beads, and magnetic beads could also be used. Wherenecessary and appropriate, lipoparticles could be engineered to containsurface molecules that specifically interact with bead coupling groups.Bead-coupled lipoparticles could be used for binding analyses such asscintillation proximity assays and immunobinding assays, affinitychromatography and protein purification applications, flow cytometry,and multiplexed binding assays.

Example 113 Co-Localization of Multiple Reporters within Lipoparticles

Carboxy-SNARF1 (Molecular Probes) is a pH-sensitive dye reporter with anexcitation wavelength of 488 nm. Its emission spectrum undergoes apH-dependent shift, and is typically monitored at two wavelengths, 580nm and 640 nm. Alexa-fluor 488 (Molecular Probes) is a general-purpose,pH-insensitive, photostable fluorescein substitute with an absorptionmaximum at 488 nm, and an emission spectrum that is typically detectedat 520 nm. These two reporters are simultaneously incorporated intolipoparticles. Lipoparticles containing a Gag/carboxylesterase andsurface biotinylation, both of which are described herein, are produced.Carboxy-SNARF1-AM acetate is incorporated by incubating the dye with thelipoparticles, wherein the esterase cleaves the AM-ester bond and trapthe SNARF1. An Alexa-fluor 488-biotin conjugate is linked to the surfaceof the lipoparticles. Unbound or unincorporated molecules are removed bypassing lipoparticles through sucrose cushions, and lipoparticlefluorescence (at 520 nm, 580 nm and 640 nm) assessed by microscopy andin a stirred-cuvette fluorometer. One skilled in the art would recognizethat a variety of reporter dyes and proteins could be incorporated intolipoparticles in combination using these, or alternative, methods.

Example 114 Incorporation of an Antibody-Anchoring ZZ-TM Fusion Proteininto Lipoparticles for Targeting

The targeting component comprises a variable target-recognition domain(an antibody), an anchor for fixation to the lipoparticle surface (a1-TM protein domain), and a domain to link these two functional units(an antibody-binding protein, fused to the 1-TM anchor). The Tva-ZZfusion protein (courtesy of Dr. Paul Bates) is a 1-TM sensor comprisingthe following contiguous domains (from amino- to carboxy-termini): aleader sequence, two Staphylococcal protein A Z-domains, and thetransmembrane domain of the single transmembrane protein Tva. Whenexpressed within the membrane of a lipoparticle, the exterior Z-domainsof the ZZ-TM fusion are capable of ‘capturing’ antibody by binding theirconstant (Fc) domains, and expressing them in the correct orientationfor target recognition and binding.

Lipoparticles containing Tva-ZZ were produced in HEK-293 cells usingmethods previously described. The presence of Tva-ZZ in Lipoparticleswas verified by binding of fluorescent antibodies to the lipoparticlesand visualizing the fluorescently-labeled lipoparticles by microscopy(FIG. 26). The incorporation of Tva-ZZ was also confirmed by VELISA,capturing Tva-ZZ lipoparticles onto wells coated with an antibody anddetecting the presence of Gag.

One skilled in the art would recognize that alternative antibody-bindingdomains (e.g. Protein G, Protein L), or 1-TM anchoring backbones (e.g.CD4, PDGFR, EGFR), could also be used to create the ZZ-TM recognitionprotein. One skilled in the art would recognize that additional methodsof incorporating functional membrane protein anchors into thelipoparticles are also possible, including avidin-biotin linkage, aminecoupling, genetic fusion of Fv fragments onto reporters, orincorporation of naturally expressed 1-TM receptors. One skilled in theart would recognize that the recognition domain of the ZZ-TM targetingcomponent could comprise any monoclonal or polyclonal antibody, and thatthe target specificity could be defined simply by changing theepitope-recognition characteristics of the ‘captured’ antibody. Oneskilled in the art would recognize that the bead could be made tocontain any number of molecules on its surface, including antibodies,antibody fragments, fluorescent molecules, peptides, proteins, smallmolecules, or organic compounds, that may be complementary to membraneproteins on the lipoparticle. Alternative target recognition domains,including ligand-specific binding proteins, could also be linked to amembrane anchor in a similar manner.

Example 115 Incorporation of Modifying Proteins to ProvideMicro-Environmental Sensitivity of Lipoparticle Probes

The goal of this experiment is to co-incorporate a modifying component,the membrane protein ion channel, TRPV1, into the lipoparticle membrane,and the calcium-sensitive reporter, FURA-2, into the lipoparticleinterior. The Transient Receptor Potential Channel V1 (also known as thevanilloid receptor 1 (VR1)), is a 95 kDa Ca⁺⁺-ion channel that opens inresponse to heat and binding of appropriate ligands (capsaicin,resiniferatoxin, and anandamide) (Caterina, et al. (1997), Nature,389:816-24, Clapham (2003), Nature, 426:517-24). Fura-2 is one of themost commonly used fluorescent reporters of calcium ions within livingcells. On binding Ca⁺⁺ (Kd 0.14 μM), Fura-2 undergoes a change in itsexcitation spectrum that can be detected either as an increase influorescence or, most commonly, as an increase in the 340/380 nmexcitation ratio (Molecular Probes Handbook (2003)). Lipoparticlescontaining the TRPV1 modifying component undergo selective and activeCa⁺⁺ internalization upon channel activation (by heat or ligand). TheFURA-2 signaling component in the lipoparticle interior allows theselipoparticles to act as caged sensors of microenvironmental calciumflux. Lipoparticles containing an esterase are produced from aGag/carboxylesterase fusion protein. A Fura-2-AM ester is incorporatedinto the TRPV1-lipoparticles where it is trapped after removal of the AMester group by the action of the Gag/carboxylesterase. UnincorporatedFura-2 AM is removed by passing lipoparticles through sucrose cushions.Aliquots of these lipoparticles are suspended in buffered solutionscontaining various concentrations of calcium. TRPV1 is activated byheating to 43° C. and/or by addition of a TRPV1 ligand (e.g. capsaicin),and fluorescence monitored by microscopy and in a stirred-cuvettefluorometer. One skilled in the art would recognize that alternativecalcium-sensitive reporters, such as the bioluminescentcalcium-sensitive enzyme, aequorin, could be included in lipoparticles.Other ion channels responding to other desired stimuli, or alternativemodifying proteins (such as surface enzymes) could also be included. Avariety of reporters, such as pH sensitive dyes, or membrane-potentialreporters, could also be co-integrated to create lipoparticle probes.Any number of these probes, either individually or in combinations,could be incorporated into lipoparticles.

Example 116 Use of Lipoparticles as Receptor-Directed ImmunofluorescentProbes

Lipoparticles containing the CXCR4 membrane protein were produced usinga Gag/GFP fusion protein. Separately, ProA beads were mixed with eitheran anti-CXCR4 antibody (recognizes a conformational domain on theextracellular side of the receptor), an anti-FLAG antibody (negativecontrol), or no antibody at all. An aliquot of the CXCR4GFP-lipoparticles were mixed with the beads, non-bound probes removed bywashing, and the beads visualized by fluorescent microscopy. Resultsindicate that the lipoparticles bound only to the beads with theanti-CXCR4 antibody (FIG. 27). Minimal or no staining of the beads wasobserved with the negative control antibody (FLAG) and with the noantibody control.

One skilled in the art would recognize that a variety of alternativefluorescent or non-fluorescent reporters could have been used.Similarly, lipoparticle probes could be used to detect proteins in othercell and tissue sample formats, such as other adherent cell cultures orcell suspensions, in paraformaldehyde or other processed tissuesections, or in tissue explants. Lipoparticle probes could also be usedto detect targets using other formats such as flow cytometry, ELISA, andwestern blot analysis (far western).

Example 117 Use of Lipoparticles as Antibody-Directed ImmunofluorescentProbes

Lipoparticles can be used as antibody-directed Immunofluorescent probes.Lipoparticles containing a ZZ-TM targeting fusion protein are producedusing a Gag/GFP fusion protein. These lipoparticles are madetarget-specific by ZZ-TM binding of a monoclonal antibody (12G5) thatrecognizes the extracellular domain of the G protein-coupled receptorCXCR4. CXCR4 is transiently expressed in HEK-293 cells using standardtransfection techniques. 24-48 h post-transfection, an aliquot of theCXCR4-specific GFP-lipoparticles is added to the culture, non-boundprobes removed by washing, and the cells visualized by fluorescentmicroscopy. Negative controls will include lipoparticles expressingirrelevant antibodies, lipoparticles without ZZ-TM, and cells notexpressing CXCR4.

One skilled in the art would recognize that a variety of alternativefluorescent or non-fluorescent reporters could be included, and thatother cell or tissue proteins could be detected simply by altering theantibody captured by the ZZ-TM targeting protein. Similarly,lipoparticle probes could be used to detect proteins in other cell andtissue sample formats, such as other adherent cell cultures or cellsuspensions, in paraformaldehyde or other processed tissue sections, orin tissue explants. Lipoparticle probes could also be used to detecttargets using other immunochemistry-based detection modalities such asflow cytometry, ELISA, and western blot analysis (far western). Thetargeting membrane protein on the lipoparticle may be an antibody boundto an incorporated ZZ-TM protein, or could be a cellular membraneprotein.

Example 118 Detection of pH Using a Lipoparticle Probe

Fluorescent dyes provide increased sensitivity for optical pHdetermination compared with conventional pH-sensitive dyes, and theincorporation of fluorescent dyes into Lipoparticles confers advantagesof spatial localization when compared with electrode-based techniques.Lipoparticles are produced and the alkylated pH-sensitive bluefluorescent dye, 4-heptadecyl-7-hydroxycoumarin (pKa range from6.35-11.15 depending on the ionic composition of the membrane) isallowed to partition into lipoparticle membranes, as describedpreviously. Fluorescence is measured in a stirred-cuvette fluorometerunder pH conditions ranging from pH 4-12. Similarly, fluorescence canalso be measured using a microscope under epifluorescent illuminationand while changing the pH in the solution above the lipoparticles.

One skilled in the art would recognize that alternative pH-sensitivedyes, such as fluorescein, FITC, Oregon Green, SNARF-1/AM, and SNAFLcould similarly be loaded into Lipoparticle membranes by this, oralternative incorporation methods. Reactive pH-sensitive dyes could beincorporated into lipoparticles by conjugation to other lipophilicmolecules such as phospholipids, or by reactive processes such asesterification or covalent attachment to Lipoparticle components.AM-esters could be allowed to diffuse directly across lipoparticlemembranes, while pH-sensitive proteins, such as PHFluorin or other pHsensitive GFP-modifications (Hanson, et al. (2002), Biochemistry,41:15477-88), could be incorporated into lipoparticles by fusion to Gagor a membrane protein as previously described.

By incorporating alternative dyes or reporters into them, Lipoparticlescapable of detecting a variety of environmental conditions can becreated for use in cells and tissues as well as in biological andenvironmental samples. The Molecular Probes Handbook (Haugland (2003))details the properties of thousands of these reporters and indicators,all of which are incorporated by reference herein. For example, calciumions can be detected using Fura-2, Indo-1, Fluo-3, Rhod-2, Fura-C18,Fura-FF-C18, Calcium Green, Calcium Orange, Oregon Greens, FFP18(Gomez-Reino, et al. (1999), Arthritis & Rheumitism, 42:989-992), andaequorin protein. Magnesium can be detected by incorporating mag-indo-1or mag-fura-2. Chloride ions can be detected using indicators such asSPQ, MQAE, MEQ, and L-6868, while sodium and potassium ions can bedetected using such reporters as SBFI and PBFI, both of which areavailable as AM-esters, or the membrane-permeant Sodium Greentetra-acetate. Other metal ions can be detected by incorporatingFluoZin-1 or FuraZin-1 (Zn2+), Phen Green FL (Cu+2, Fe, Hg, Pb, Cd, Ni),NewportGreen (Ni+2, Zn), while rhodamine 6G can be used to detecthypohalites (ClO—). Chelators and ionophores such as nitrophenyl EGTA,DMNP-EDTA, Diazo-2, BAPTA-AM, TPEN (chelates Zn, Cu, Fe), or theionophore 4-Bromo A-23187) can also be incorporated into Lipoparticles.Environmental contaminants could also be detected by incorporation ofappropriate reporters. For example, thiols and cyanide can be detectedby the fluorescence of CBQCA. A variety of agents capable of detectingreactive oxygen species are available, including M-7913, MCLA,dihydrocalcein-AM, malachite green, Amplex Red,diphenyl-1-pyrenylphosphosine (DPPP) and coelenterazine, and nitricoxides can be detected by DAF-FM, SNAP, 2,3-diaminoapthalene, and SBDmethylhydrazine. Oxidation and reduction can be monitored using agentssuch as resazurin, dodecyl resazurin, dihydrorhodamine, ordihydrofluoresceins. Any of these dyes can be incorporated intolipoparticles individually or in combination.

Such reporters could be incorporated into Lipoparticles by any of themethods outlined previously, or using combinations of these methods.These include, but are not limited to, fusion to Gag-, membrane-, orother lipoparticle proteins, partitioning of lipophilic dyes orlipid-conjugates within the membrane, encapsulation within thelipoparticle by diffusion of AM-esters or lipoparticle poration orpermeabilization, and linkage or covalent attachment to the lipoparticlesurface.

Example 119 Microinjection of Lipoparticles into Cells

Intracellular Lipoparticles bearing a variety of targeting and signalingcomponents are capable of localizing desired subcellular structures andevents, and reporting them through emission of detectable luminescent orfluorescent signals. The compartmentalization of functional componentswithin the lipoparticle vehicle enables the Lipoparticle to contain andlocalize reporters to target sites and minimize non-specific backgroundemissions, to limit general disruption to the cytoplasmic equilibrium,and to utilize high concentrations of potentially toxic reporters (e.g.ion chelators). Lipoparticles bearing user-specified effector proteinsand other modifying components (e.g. ion channels, enzymes) are capableof selective and specific delivery of active molecules to subcellularlocations, and of microenvironmental perturbation.

Labeled lipoparticles are prepared by producing lipoparticles using aGag/GFP fusion protein as outlined herein, and are suspended inCa⁺⁺-free HBS. HEK-293 cells, grown to confluence, are microinjected toapproximately one-tenth of the cell volume with an aliquot of Gag-GFPLipoparticles, using a back-loaded glass needle controlled with aNarshige micromanipulator. Placement is monitored before, during andafter the procedure (up to two hours) by direct microscopicvisualization using epifluorescent illumination. One skilled in the artwould recognize that a variety of alternative placement techniques, suchas those utilizing biolistics, endocytosis pathways, and proteintransfection, could also be used to introduce Lipoparticles into cells.

Example 120 Detection of Cytosolic Fluctuations in Ca⁺⁺ ConcentrationUsing Lipoparticles

Lipoparticles are constructed by producing TRPV1-containinglipoparticles using a Gag/carboxylesterase-fusion protein as outlinedherein, suspending them in Ca⁺⁺-free HBS, and loading them with the Ca⁺⁺reporter, FURA-2. HEK-293 cells are grown to confluence, transferred tocoverslips, and allowed to adhere. Lipoparticles are microinjected into20 cells per coverslip, as described herein. Cells are stimulated withheat (heating to 43° C. activates TRPV1 in the Lipoparticles) andtreated with TRAP-6, an agonist of the thrombin G-protein coupledreceptor. Activation of the thrombin receptor initiates Ca⁺⁺ flux withinthe cell, causing Ca⁺⁺ entry into Lipoparticles through the open TRPV1channels. Interaction of Ca⁺⁺ with the trapped Lipoparticle reporter,FURA-2, causes fluorescent emission, which is recorded in real timeusing a CCD camera mounted on a Nikon TE2000 inverted microscope using a100× objective.

One skilled in the art would recognize that alternative means ofactivating TRPV1, including the use of ligands such as capsaicin,resiniferatoxin, and anandamide, and that alternative methods ofstimulating this, and other cell signaling pathways, such as forskolintreatment, could similarly be used. A variety of cytoplasmic structures(e.g. cytoskeleton, membrane structures, transcription machinery) andevents (e.g. cell signaling, enzyme activity, change in membranepotential, transcription and translation) could be monitored by alteringthe targeting, signaling and modifying components of the Lipoparticleappropriately. For example, the inclusion of the antibody-based ZZ-TMtargeting component could localize and confine Lipoparticles to specificsubcellular locations or events. The simultaneous combination of varioustargeting and signaling components could allow for the monitoring ofcytoplasmic Lipoparticles using multimodal detection systems. Othermodifications to the lipoparticle vehicle or components, such asPEGylation or polymerization of lipids, and fixation of proteins, couldenhance or alter LipoProbe function within cells. Lipoparticles couldalso be used with a diverse range of other target cell formats, such asalternative secondary cell culture lines, primary cell cultures orisolates, permeabilized cells, and tissue explants or sections.

Example 121 Use of Lipoparticles as Modified Labeled Viruses

The purified virus is biotinylated and linked to streptavidin quantumdots, as described herein. Macrophages are prepared from healthy humanvolunteers using standard methods of practice. Labeled HIV is layeredover the cells, and the interaction of the virus with the macrophagesmonitored in real time using a fluorescent microscope at 100×magnification and using a warmed microscope slide stage. Lipoparticlescontaining quantum dots but not expressing HIV Env are used as negativecontrols.

Labeled HIV virus can also be followed in vivo. A suspension of labeledHIV (containing quantum dots) in 50 μl of sterile HBS is injectedintravenously into female CD1 mice (8 weeks of age). Mice are euthanized24 hours later, conventional blood smears made, and histologicalsections of liver, spleen, kidney, lungs, lymph nodes, intestine, andbrain prepared, and examined using fluorescent microscopy. Data areexpressed as the proportion of tissue and cell populations exhibitingthe quantum dot markers. Virions are expected to migrate to a number oftissues and cell types, including macrophages, dendritic cells, lymphnodes, and the liver. Toxicity, immune response, and the ability totarget desired tissues and cells form the criteria for assessing theperformance of Lipoparticles in live animals.

One skilled in the art would recognize that Lipoparticles expressing avariety of viral (e.g. MLV Envelope) and non-viral membrane proteins(e.g. EGFR, Fas-ligand, TNF receptor, Integrins), and containing avariety of reporters could be used ex vivo (with isolated cells ortissues) or in vivo in a similar manner. Lipoparticles could also beused to monitor protein and other active molecular interactions withprimary or immortalized cells in culture. Lipoparticles could be used tomonitor the effect of isolated viral or cell components on cellfunctions such as processing and phagocytosis in macrophages, or onwhole-organism processes such as viral migration and tissue trafficking.These measurements could be made in real time, in whole animals (in vivoimaging), or as end-point measurements.

Example 122 Method of Hybridizing a Nucleic Acid Probe to a Lipoparticle

A large variety of fluorescent dyes that are capable of binding nucleicacids are available commercially. These dyes interact with features ofDNA or RNA superstructure (e.g. SYBR green and DAPI bind the minorgroove of double-stranded DNA) or with the nucleotide bases (e.g. YoYo1and propidium iodide interchelate between the bases). These dyes arecommonly used to monitor cell cycle activity in mammalian cells, and canbe used to label nucleic acids within viruses and viral-derivedstructures such as lipoparticles. Retroviral structures that do notincorporate their own genomes will incorporate host cell RNA instead.

Lipoparticles were produced from HEK-293 cells using methods previouslydescribed, and suspended in 10 nM solutions of YOYO-1 in HBS. After 20min, Lipoparticles were visualized using fluorescence microscopy (FIG.28). The stained lipoparticles could be visualized as fluorescent spots.One skilled in the art would recognize that alternative nucleicacid-specific fluorescent dyes could also be used, including, but notlimited to, SYBR green, PicoGreen, SYTOX-1, ethidium bromide, DAPI,YO-PRO-1, TOTO-1, POPO-3, and propidium iodide. The lipoparticle may bestained with such dyes either before or after permeabilization withagents such as detergents or pore-forming peptides, or by mechanicalmeans such as electroporation or sonication.

Example 123 Altering the Biochemistry of the Lipoparticle Interior

Lipoparticles can be used to monitor micro-environmental conditions,such as ion concentration, utilizing such sensing and modifyingcomponents as membrane ion channels, and to deliver molecules tospecified extra- or intra-cellular sites. The efficiency and sensitivityof these Lipoparticle functions may be modified by characteristics ofthe lipoparticle contents, such as ion concentration, which are, to someextent, user-definable. The ionic content of lipoparticles can bealtered by treating them with appropriate aqueous solutions. Althoughthe lipid bilayer of lipoparticles is generally considered to beimpermeable, ions will equilibrate across it over time. By soakinglipoparticles in solutions of high ion concentration, the ion content ofthe lipoparticles can be increased, and conversely, it can be decreasedby soaking lipoparticles in solutions of low ion concentration.Lipoparticles (1 μg) are suspended in HBS containing 150 mM CaCl₂ andstored in darkness at 4° C. At 24 hour intervals, aliquots are passedthrough G50 Sephadex columns and re-suspended in HBS without CaCl₂.Lipoparticle fluorescence (indicating interior Ca⁺² accumulation) ismeasured in a stirred cuvette fluorometer by adding calcein, afluorescent dye that fluoresces brightly in the presence of calcium.Lipoparticles without added CaCl₂ are used as a negative control. Oneskilled in the art would recognize that other biochemicalcharacteristics of the lipoparticle interior, such as the concentrationof other ions or small molecules, could also be altered by similarmeans.

Example 124 Stabilizing Lipoparticle Structure

Fixation of complex biological structures, such as cells and tissues,with aldehyde solutions such as formaldehyde and paraformaldehyde,results from the formation of methylene bridges between protein nitrogenatoms. This cross-linking preserves protein structural integrity, andforms an insoluble matrix that traps carbohydrates and lipids withoutaltering their chemical composition. Fixation of lipoparticle protein ormembrane constituents can alter their longevity and behavior in a numberof applications such as immuno-probing. Lipoparticles containing thechemokine receptor CXCR4 are constructed by previously describedmethods. Aliquots of CXCR4-lipoparticles (1 μg total protein) aresuspended in a 5% solution of paraformaldehyde in HBS, and aldehydebinding is allowed to proceed for 24 hours. Lipoparticles are thensoaked in HBS, with several changes of solution, for 24 hours to removeunbound aldehyde molecules. Lipoparticles are passed through sucrosecushions and resuspended in fresh HBS. The structural integrity of themembrane proteins is verified by VELISA using conformationally-dependentMAbs, as described herein. The ability of CXCR4 to bind its cognateligand, SDF-1, is also assessed by biosensor analysis. Unfixedlipoparticles containing CXCR4, and lipoparticles containing no specificmembrane protein are used as controls. One skilled in the art wouldrecognize that fixation of other lipoparticle membrane or non-membraneprotein constituents could also be achieved using the same procedure,and that alterations to the fixation conditions, such as time,temperature, or buffer composition, could produce essentially similarresults. Other fixatives, such as alternative protein cross-linkingaldehydes (e.g. glutaraldehyde), or lipid fixatives (e.g. osmiumtetroxide) could also be used to stabilize lipoparticle structuralconstituents.

Example 125 Incorporation of Activating Molecules

Lipoparticles can be constructed to be capable of selective delivery ofan active molecule, ryanodine, to intracellular ryanodine receptors. Theryanodine receptor (RyR) is a multi-isoform, intracellular membrane Ca⁺⁺channel responsible for regulation of cytoplasmic calcium concentrationin a variety of cells, including muscle cells (where it is found in thesarcoplasmic reticulum), and brain cells. Ryanodine, an ester ofpyrrole-α-carboxylic acid with ryandolol, is a plant alkaloid capable ofbinding to and altering the activity of RyRs. Lipoparticles containingthe ZZ-TM targeting fusion protein are constructed by methods describedherein. Aliquots of ZZ-TM Lipoparticles are re-suspended in HBScontaining 1 mmol/L of BODIPY-FL-X ryanodine (Molecular Probes),electroporated into lipoparticles. Unincorporated ryanodine is removedby passing lipoparticles through a Sephadex G50 spin column,resuspending in HBS. Lipoparticle fluorescence is measured in a stirredcuvette fluorometer, and monitored by direct visualization usingfluorescence microscopy. FL-Ryanodine-ZZ-TM lipoparticles are madetarget specific using a monoclonal antibody (RDI-RYANRabm; ResearchDiagnostics Inc.) that recognizes RyR-1 and RyR-2 in a broad range ofspecies and tissue types. HEK-293 cells are grown to confluence,transferred to coverslips, and allowed to adhere. Lipoparticles aremicroinjected into 20 cells per coverslip, and their intracellulardistribution monitored in real time by direct visualization usingfluorescent microscopy. Lipoparticles not containing a targetingantibody are used as controls. RyRs will be co-localized by doubleimmunohistochemistry using a primary monoclonal antibody for RyR, and afluorescently-labeled secondary antibody.

One skilled in the art would recognize that alternative methods forloading FL-ryanodine or other active compounds into Lipoparticles couldalso be used. One skilled in the art would recognize that a largevariety of active compounds (e.g. terbium, bisindolylmaleimide,polymixin B, calmodulin, P2X receptor agonists (BzBzGTP and BzBzATP)),either native or modified (e.g. fluorescently tagged, caged), couldalternatively be incorporated into the lipoparticle interior, or ontothe lipoparticle surface, by this, or other methods. Using similarstrategies, substrates can also be incorporated into lipoparticles, suchas ELF97 (a fluorescent substrate), caspase-3 peptide substrates, DiFMUPphosphatase substrate, or other substrates that change color and/orfluorescence when altered by an enzyme such as beta galatosidase, aprotease, a phospholipase, a kinase, or that respond to thiols,phosphates, pyrophosphates, or free phosphate, could also beincorporated into lipoparticles (Haugland (2003)). Lipoparticles couldbe used for the selective intracellular delivery of active molecules toa variety of cell and tissue formats, including cultured cells, tissuesections or explants, and in vivo.

Example 126 Detection of Lipoparticle-Virus Fusion

Lipoparticles can be used to monitor the process of receptor-mediatedviral fusion using Lipoparticles containing viral envelope proteins(“effectors”) and Lipoparticles containing viral receptors (“targets”).Gag/CFP-Lipoparticles containing the murine leukemia virus envelopeprotein (MLV-Env) are constructed. Separately, Gag/YFP-Lipoparticlescontaining murine Tva, a member of the LDL-superfamily of membraneproteins and a cognate host cell receptor for MLV, are also constructed.Gag/CFP and Gag/YFP are capable of fluorescence resonant energy transfer(FRET) if the fluorophores come into close proximity (<50 Å). This ispossible in a mixing assay only if Lipoparticle membranes fuse as aresult of MLV-Env interaction with Tva. Aliquots of each Lipoparticlepreparation are mixed in HBS, and fluorescence is monitored in astirred-cuvette fluorometer. When the particles fuse, FRET can bemeasured. Lipoparticles prepared with both Gag/CFP and Gag/YFP in thesame particle are used as positive controls. Gag/CFP and Gag/YFPLipoparticles lacking either or both membrane proteins are used asnegative controls.

One skilled in the art would recognize that a variety of alternativemethods for detecting the mixing of Lipoparticle contents can also beused to indicate fusion of lipoparticles. These include, but are notlimited to, alternative FRET pairs (e.g. rhodamine and NBD, BODIPY andrhodamine, BODIPY and Texas Red), BRET pairs, dye/quencher pairs (e.g.ANTS/DPX and Tb³⁺/DPA), dye/substrate pairs (e.g. Fluo-3 and calcium),and enzyme/fluorescent substrate pairs (e.g. carboxylesterase andcalcein-AM). Alternatively, lipoparticle fusion can be detected byincluding reporters that indicate the mixing of membrane lipids. Forexample, FRET can occur from mixture of a donor phospholipid conjugate(e.g. NBD-PE) with an acceptor phospholipid conjugate (e.g.Rhodamine-PE). Other examples of detection systems can include octadecylrhodamine B self-quenching and pyrene excimer formation, as described inthe Molecular Probes handbook (Haugland (2003)). The viral-host cellinteraction to be studied can be varied simply by including the desiredviral and mammalian protein targets/targeting components. The system canbe used to identify previously unknown viral host cell receptors, tomonitor viral-host cell interaction kinetics, or to screen potentialpharmaceutical inhibitors of viral-cell fusion.

Example 127 Detection of Lipoparticle Binding to Cells by FRET

FRET technology can be combined with the lipoparticle for detection ofbinding of the lipoparticle containing one receptor to cells containinga complementary receptor. Lipoparticles containing the membrane proteinHIV Envelope are labeled with the fluorescent phospholipid rhodamine-PE.Separately, HEK-293 cells expressing CD4 on their surface are labeledwith a phospholipid conjugated to the NBD fluorophore. When labeledparticles are mixed with labeled cells, FRET occurs only when Env bindsCD4. FRET is detected in using a Wallac Victor2V with samples in 96-wellmicroplates. Labeled lipoparticles without Env and similarly labeledcells without CD4 can serve as negative controls. Such a system can beused to screen a library of candidate molecules for inhibitors of themembrane protein interactions.

One skilled in the art would recognize that the lipoparticle used herecould be a live, replication-competent virus isolated from cells or apseudotyped virus containing a viral Envelope protein. Similarly, thecells could be composed of cell lines, primary cells, or isolatedtissues. On skilled in the art would also recognize that multipleadditional fluorophores that serve as FRET pairs could also be used,including lipophilic dyes that can come into close proximity uponbinding of lipoparticles.

Example 128 Detection of Ligand Binding to Lipoparticles by FRET

FRET technology can be used with the lipoparticle for detection ofbinding of a ligand to a receptor on the lipoparticle membrane.Lipoparticles containing the GPCR CXCR4 are labeled with the fluorescentphospholipid rhodamine-PE. Separately, the chemokine SDF is labeled withan NBD fluorophore. When labeled SDF is mixed with labeledlipoparticles, FRET will occur only when SDF binds to CXCR4. Labeledlipoparticles without CXCR4 and similarly labeled but non-specificchemokines can serve as negative controls. FRET is detected in using aWallac Victor2V with samples in 96-well microplates. Such a system canbe used to screen a library of candidate molecules for inhibitors of theligand-receptor interaction. One skilled in the art would recognize thatmultiple additional fluorophores that serve as FRET pairs could also beused.

Example 129 Detection of Ligand Binding by Polarization

Lipoparticles can be used to detect binding of fluorophore-coupledHOE140 to lipoparticles expressing B2 bradykinin receptors by measuringfluorescent emission polarity. When fluorophores are exposed topolarized light such as a laser, molecules with their absorptiontransition vectors aligned parallel to the excitation vector will beselectively excited. During the period between excitation and emission,each fluorescent molecule will rotate such that light emission vectorswill become randomized. As the speed of this molecular rotation isdependent upon the molecular weight of the fluorescent particle,fluorophores with lower molecular weights will be characterized by amore highly randomized emission vector distribution than those withhigher molecular weights. A shift in the fluorescent emission polaritycan be exploited to monitor the binding of relatively low molecularweight, fluorescently-labeled ligands (such as peptides and steroids) torelatively high molecular weight receptors or lipoparticles containingthese receptors (the shift in polarity associated with the sizedifferential between ligands and receptors expressed in lipoparticleswould be extreme). Lipoparticles containing the B2 bradykinin receptor,a G-protein coupled receptor, are produced in HEK-293 cells. Aliquots ofthe B2-lipoparticles are suspended in HBS, added to the wells of 96-wellplates and BODIPY TMR dye-labeled HOE140 (Molecular Probes) added inincreasing concentrations. BODIPY dyes generally interfere less withreceptor-binding affinity, possess greater molecular-weight rangepolarization sensitivity, and are less influenced by intrinsic samplefluorescence than other, more conventional dyes such as fluorescein andrhodamine. Fluorescence polarity is measured in real time using a WallacVictor2V fluorescence plate reader (Perkin Elmer). Lipoparticles notcontaining any specific receptor are used as negative controls.

One skilled in the art would recognize that various other fluorescentlabels can also be used as alternatives to BODIPY, and that a variety ofligand-receptor interactions can be similarly monitored by varying theligand and the receptor incorporated into the lipoparticle. The bindingof unlabeled ligands can similarly be monitored by adding a soluble,labeled ligand binder. The assay can be used to monitor binding kineticsof ligand-receptor interactions in the presence or absence of potentialinhibitors, such as in high-throughput drug screening applications.

Example 130 Detection of Ligand Binding by Microscopy

Lipoparticles expressing the G-protein-coupled melanocortin receptor,MC4, are prepared. MC4-lipoparticles are immobilized on glass slides byadsorption, and non-specific adsorption sites blocked for 20 minutesusing a 1% solution of BSA in HBS. Individual slides are then exposed toHBS solutions containing increasing concentrations of fluorescentlylabeled Fluo-NDP-αMSH (a fluorescein-labeled analog of the 13-mer MC4agonist NDP-αMSH; Advanced Bioconcept Co.). After 45 minutes, slides arewashed several times with buffer, and imaged at 100× magnification on afluorescent microscope. Fluorescence intensity is quantified fromdigital images using Canvas software. Lipoparticles not containing anyspecific receptor are used as negative controls.

Additional fluorescent molecules, listed in the Molecular ProbesHandbook (Haugland (2003)) and incorporated by reference herein couldalso be used. Such molecules include fibrinogen, gelatin, Type IVcollagen, casein, cytochalasin B, Lipopolysaccharide (LPS), endostatin,fMLF receptor peptide, alpha-MSH (Melanocyte stimulating hormone)peptide, dexamethasone, Low-density lipoprotein (LDL), epidermal growthfactor (EGF), transferrin, lactoferrin, fibrinogen, ovalbumin, bovineserum albumin, soybean trypsin inhibitor, Histone H, alpha crystalline,hyaluronic acid, mucin, subunit B of cholera toxin, chemotacticpeptides, insulin, and heparin. Fluorescent molecules also includeprobes that bind to ion channels such as the L-type Ca+2 channel,intracellular Ca-channels, calcium pump, Na+/H+antiporter, Na+ channel,Na/K+ ATPase, Ca+-activated K+ channel, ATP-dependent K-channel,Glutamate gated Cl-channel. Fluorescent molecules also include probesthat bind to neurotransmitter receptors such as alpha-Bungarotoxin(Nicotinic ACHR), acetylcholinesterase substrate, muscarinicacetylcholine receptor, pirenzepine fluorescent antagonist, prazosin(alpha1-adrenergic receptor), CGP 12177 (beta-adrenergic receptor), andmuscimol (GABA-A receptor). Fluorescent molecules also includeneuropeptides such as substance P, neuromedin C, angiotensin II,Naloxone, and naltrexone.

One skilled in the art would recognize that additional fluorescent orluminescent tags could be used as alternatives to fluorescein, and thatthe binding of ligands to a variety of receptors could be monitored in asimilar manner by varying the receptor content of the immobilizedlipoparticles. The use of confocal microscopy could overcome the need towash slides, and allow monitoring of ligand to receptors in real time.The use of TIRF microscopy or flow cells could improve sensitivity.Alternative formats such as microfluidic flow cells and plates couldalso be used.

Example 131 Detection of Lipoparticles by Flow Cytometry

Flow cytometry is a technology in which simultaneous measurements ofmultiple characteristics are made on individual objects (such as cellsor beads). A fluidics system manipulates the objects for interrogation,an optics system generates and collects light emissions, and anelectronics component records the optical signals into digitalrecordings for analysis. Although some parameters of flow cytometry canbe limited by resolution, other characteristics, such as side scatterand fluorescence intensity, can be measured for objects <200 nm.Lipoparticles containing a fluorescent fusion membrane protein,CXCR4/GFP, were produced and suspended in HBS. The Lipoparticlesuspension was analyzed using a FACSCalibur flow cytometer (BDBiosciences), and gated using side scatter and fluorescent emissionintensity (530 nm for the Lipoparticle gating reporter, GFP, afterexcitation at 488 nm). Lipoparticles containing a non-fluorescentversion of CXCR4 (Lestr-HA) were used as a negative control. Resultsanalyzed with CellQuest software indicate that fluorescent lipoparticlescould be easily distinguished from non-fluorescent lipoparticles by flowcytometry (FIG. 29). 200 nm Fluoresbrite YG beads (Polysciences) werealso used as a positive control.

One skilled in the art would recognize that a variety of reporters,incorporated by a number of methods, as described herein, could also beused to detect and identify lipoparticles for gating. These includefluorescent proteins fused to the structural core protein Gag, or tomembrane proteins within the Lipoparticle, lipid-soluble fluorescentdyes, AM-ester dyes, or fluorescent dyes conjugated to the Lipoparticlesurface using streptavidin-biotin linkage or covalent attachment. Oneskilled in the art would also realize that the number of fluorescentevents counted by the flow cytometer is directly proportional to thenumber of lipoparticles in the sample, providing an estimate of thenumber of lipoparticles in the sample.

Example 132 Detection of Ligand Binding by Flow Cytometry

Commercial flow cytometers, in addition to detecting light diffraction(forward and side scatter) as an indication of size and internalgranularity, are capable of making multiple, simultaneous measurementsof fluorescent light emission intensity. Lipoparticles are constructedby producing lipoparticles containing CXCR4 using a Gag/GFP fusionprotein as described herein. Aliquots of CXCR4-Gag/GFP-Lipoparticles (1μg of protein) are suspended in HBS and 100 ng-100 ug solutions ofAPC-conjugated 12G5 antibody (a monoclonal antibody recognizing aconformational epitope on CXCR4) is added. After 30 minutes at roomtemperature, the Lipoparticle suspensions are passed through aFACSCalibur flow cytometer (BD Biosciences), and gated usingfluorescence emission in FL1 (530 nm, for the Lipoparticle gatingreporter, GFP) and side scatter. Fluorescence intensity in FL2 (661 nmfor the APC-conjugated antibody) is measured. Gag/GFP-Lipoparticlescontaining a ZZ-TM antibody-binding protein with captured APC-antibodyis used as a positive control. Lipoparticles not containing CXCR4 andunconjugated 12G5 antibody are used as negative controls. The specificbinding of the antibody 12G5 to the receptor CXCR4 on the lipoparticleis detected.

One skilled in the art would recognize that Lipoparticles containingeither no or alternative gating reporters (e.g. other Gag- or membraneprotein-fusions, lipophilic dyes, other dyes and reporters encapsulatedwithin the lipoparticle interior, or captured on the lipoparticlesurface) could similarly be used. A wide variety of alternative targetligands (e.g. other receptor ligands, pathogen antigens, cancer markers,pathogen-specific antibodies, auto-immune antibodies) could similarly bedetected by changing the recognition characteristics of the targetingcomponent (e.g. other membrane receptors, antibody-bound ZZ-TM targetingprotein) and the target reporter. The ligand to be detected may befluorescently labeled for direct detection of binding or unlabeled andused for competition of a labeled ligand (e.g. for drug screening). Thepresence of target ligand could be expressed either semi-quantitatively(target reporter fluorescence intensity as a function of gating reporterfluorescence intensity), or quantitatively (using standard solutions ofknown target concentration). Multimodal detection is also possible bysimultaneously combining multiple compatible targeting and signaling(gating and target recognition) components within the lipoparticlevehicle. One skilled in the art would also realize that the number offluorescent events counted by the flow cytometer is directlyproportional to the number of receptors in the sample, providing anestimate of the number of receptors in the sample.

Example 133 Detection and Quantitation of Anti-Viral Antibodies in Serum

Lipoparticles can be used to detect and quantify anti-viral antibodiesin serum. HIV Env is a single-transmembrane protein that forms trimersin its functional and antigenically-correct form. The ability toidentify and distinguish antibodies against trimeric Env versusmonomeric Env would provide a better characterization of theneutralizing antibodies present in a patient's serum. Env-Lipoparticlesare produced and biotinylated and attached to streptavidin-coatedLuminex beads. Bead/lipoparticle sets are mixed with serum derived frompatients infected with the pathogen, or with serum from uninfectedcontrols, and the antibody is allowed to bind for 1 hour at roomtemperature, and then washed with PBS buffer. A fluorescent secondaryanti-human IgG antibody is added and allowed to bind for 30 minutes atroom temperature. Bead sets are washed and then flowed through aFACSCalibur flow-cytometer (Becton Dickinson), gating the fluorescentbeads (with lipoparticles attached) using side scatter andbead-incorporated fluorescence. Each bead set can be distinguished by aunique optical signature (each bead incorporates a unique set offluorescent markers). Viral-specific antibody is quantified by therelative intensity of the reporter antibody signal. Specificquantitation may be made relative to standards of known quantity, or byminimal detectable titration of the serum.

One skilled in the art would recognize that additional bead types andmeans of attachment, including alternative biotin-avidin linkagestrategies, covalent attachment, or binding to lectins, could be used.In addition, fluorescent dyes and reporters may be incorporated intolipoparticles in various ways, by associating them with the coreprotein, membrane proteins, within the membrane lipid, or encapsulatingthem within the lipoparticle interior, either in place of or in additionto bead fluorescence. When lipoparticles are labeled, the efficiency oflipoparticle conjugation to bead sets could be determined by thedetection of lipoparticle-associated dye emission. The system may beoptimized to use several distinct viral antigens, separately orsimultaneously, for the detection of stage of infection, or viralstrain, as well as for sub-typing of immunoglobulin class by the use ofreporter antibodies specific for IgG, IgM, or IgA. One skilled in theart would recognize that antibodies to other viral membrane antigens,including EBV gp340, gp84, and gp150, RSV G- or F-protein, or Influenzavirus HA or NA, could also be detected in a similar manner.

Example 134 Multiplex Detection of Respiratory Viral Infections

Influenza and respiratory syncytial virus (RSV) account for the majorityof viral respiratory tract infections, particularly in the very young.Infections are not clinically distinguishable from one another, theseasons of highest risk of infection overlap, and there is a broad rangeof overlapping clinical manifestations, necessitating laboratorytechniques for definitive diagnosis. There is currently no technique onthe market for the simultaneous detection of antibodies for thesepathogens, and the ability to characterize the serological profile of apatient presenting with suspected viral respiratory disease will lead tomore rapid and accurate diagnosis, and more rapid instigation ofappropriate therapy. Lipoparticles containing the haemagluttinin andneuraminidase influenza viral membrane glycoproteins(HANA-lipoparticles) and lipoparticles containing the G- and F-envelopeglycoproteins of RSV (GFRSV-lipoparticles) are prepared, biotinylated,and attached to optically-unique Luminex beads. Bead/lipoparticle setsare mixed with antibody-containing serum derived from human patientsinfected with either RSV or influenza (or both), washed, and then mixedwith a fluorescent secondary anti-human IgG antibody. Bead sets aregated using side scatter and the unique fluorescent signal of each beadset, which will differentiate GFRSV-lipoparticles fromHANA-lipoparticles. Viral-specific antibodies are quantitated from theintensity of the fluorescent signal from the secondary antibody in eachgate. One skilled in the art would recognize that antibodies to otherviral membrane antigens could also be detected in a similar manner.

Example 135 Detection of Serum Response to Viral-Encoded Host CellMembrane Proteins

Lipoparticles can be used to develop a methodology for the detection ofa serum response to ORF74, a GPCR encoded by human herpesvirus 8 (HHV8),and expressed on the surface of infected cells. HHV8 infection isassociated with a number of neoplastic diseases, including Kaposi'ssarcoma and pleural effusion lymphoma. There are currently a number ofmethods for the detection of both lytic and latent viral antigens,however, the ability to detect an antibody response against host-cellexpressed viral proteins will aid in the differentiation of active andongoing infection with HHV8. Lipoparticles expressing ORF74 areprepared, biotinylated, and attached to Luminex beads. Bead/lipoparticlesets are mixed with antibody-containing serum derived from humanpatients infected with HHV8, or with serum from uninfected controls,washed, and then mixed with a fluorescent secondary anti-human IgGantibody. ORF74-Lipoparticles/beads are separated and distinguishedusing flow cytometry, and ORF74-specific antibodies quantitated from theintensity of the fluorescent signal from the secondary antibody in eachgate. One skilled in the art would recognize that antibodies to otherviral-encoded membrane antigens, such as US28, UL32 and UL78 from CMV,or U12 and U51 from HH6 and HHV7, could be detected in a similar manner.

Example 136 Detection and Quantitation of Autoimmune Antibodies in Serum

Lipoparticles can be used to detect auto-antibodies to the thyrotropin(thyroid-stimulating hormone) receptor (TSHR) in serum. TSHR, thelargest of the hormone receptors, is a seven-transmembrane Gprotein-coupled glycoprotein expressed in multiple tissues.Autoantibodies directed against this protein are implicated in bothautoimmune hypothyroidism (Hashimoto thyroiditis) and autoimmunehyperthyroidism (Graves disease). The ability to routinely detect theseantibodies in serum will complement more cumbersome confirmatorydiagnostic techniques such as the measurement of radioiodine uptake.Lipoparticles incorporating TSHR are prepared, biotinylated, andattached to Luminex beads. Bead/lipoparticle sets are mixed withantibody-containing serum derived from human patients with autoimmunethyroid disease, or with healthy control serum, washed, and then mixedwith a fluorescent secondary anti-human IgG antibody.TSHR-Lipoparticles/Beads are separated and distinguished, andTSH-R-specific antibodies quantitated from the intensity of thefluorescent signal from the secondary antibody in each gate. One skilledin the art would recognize that auto-antibodies directed against othermembrane antigens, including acetylcholine receptors (myasthenia gravis)and calcium channels (Lambert-Eaton) could also be detected in a similarmanner.

Example 137 Screening Hybridomas for Monoclonal Antibody Production

Lipoparticles can be used to simultaneously screen hybridoma culturesfor the presence of monoclonal antibodies recognizing the Epstein-Barrvirus (EBV) membrane glycoproteins gp340, gp84, and gp150. Monoclonalantibody production is a laborious and time-consuming procedure. Theability to utilize structurally intact membrane proteins (as opposed tosynthetic peptides) to screen cultures will allow the more reliableisolation of conformationally-dependent antibodies. The ability tosimultaneously detect and quantitate monoclonal antibodies produced bymultiple hybridoma cultures will be a valuable time-saving feature.Hybridomas are produced as described elsewhere (Harlow, et al. (1989)),using whole, killed EBV inoculates. Three sets of lipoparticles,expressing the EBV membrane proteins gp340, gp84, and gp150 areproduced, biotinylated, and attached to optically-unique Luminex beadsets. EBV-Bead/lipoparticle sets are mixed, singly, or in combinationfor 1 hour at room temperature with supernatant from 7-day hybridomacultures, washed, and then mixed with anti-mouse fluorescent secondaryantibody. Bead sets undergo flow cytometric analysis, gating upon sidescatter and the unique fluorescent signal of each bead set, which willdifferentiate gp340-lipoparticles, gp84-lipoparticles, andgp150-lipoparticles. Monoclonal antibodies are quantitated from theintensity of the fluorescent signal from the secondary antibody in eachgate. One skilled in the art would recognize that monoclonal antibodiesdirected against other membrane proteins, including human membraneproteins, could also be detected in a similar manner.

Example 138 Mapping Epitopes on Integral Membrane Proteins

Lipoparticles can be used to develop a system for the mapping ofepitopes of the G protein-coupled receptor CCR2b. The characterizationof monoclonal antibody interactions with structurally distinct epitopesof CCR2b enables these antibodies to be used to identify functionalcomponents of the receptor, including ligand binding sites. Hybridomasand monoclonal antibodies directed against CCR2b are produced asdescribed previously. Lipoparticles incorporating wild-type andselective variants of CCR2b (containing CCR5 region substitutes (Rucker,et al. (1996), Cell, 87:437-446)) are produced, biotinylated, andattached to Luminex beads as described previously. Bead/lipoparticlesets are mixed with a monoclonal antibody directed against CCR2b,washed, and then mixed with fluorescent anti-mouse fluorescent secondaryantibody. Bead sets undergo flow cytometric analysis as previouslydescribed, gating upon side scatter and the unique fluorescent signal ofeach bead set, which will differentiate wild-type CCR2b-lipoparticles,substituted mutant CCR2b-lipoparticles, and deleted mutantCCR2b-lipoparticles. Monoclonal antibodies are quantitated from theintensity of the fluorescent signal from the secondary antibody in eachgate. One skilled in the art would recognize that monoclonal antibodyinteraction with other intact and structurally altered antigens couldalso be detected in a similar manner.

Example 139 Multiplex Detection of Unlabeled Ligands in BiologicalFluids

Lipoparticles can be used to develop a multiplex detection system forunlabeled ligands of the G protein-coupled receptor CCR5 and CCR1. CCR5binds the naturally-occurring chemokine ligands MIP1α, MIP1β and RANTESwith nanomolar affinity. Chemokine receptors characteristicallydemonstrate wide ligand promiscuity, and some of these same ligands(e.g. MIP1α) are also known to bind other chemokine receptors, forexample CCR1. The ability to detect and distinguish such natural ligandsin biological fluids would be useful for diagnostic and researchpurposes. A system that can detect ligand-receptor interaction wouldalso be useful for the identification of different, previouslyunrecognized receptor-ligand pairs. Two sets of lipoparticles areprepared, incorporating CCR5 or CCR1. Lipoparticles are attached toLuminex beads, pairing CCR5 lipoparticles with one uniquely colored beadset and CCR1 lipoparticles with a different uniquely colored bead set.Bead/lipoparticle sets are mixed with human serum for one hour, washedin Hepes buffered saline, and then mixed with fluorescently-labeledanti-MIP1α, anti-MIP1β, and anti-RANTES antibodies, each fluorescentlylabeled with a different color. Bead sets undergo flow cytometricanalysis as previously described, gating upon side scatter and theunique fluorescent signal of each bead set. The unique fluorescentpattern of each Luminex bead set differentiates CCR5- fromCCR1-containing beads. Binding of ligands to each receptor isquantitated from the intensity of the fluorescent signal from thefluorescently labeled antibody against each chemokine ligand. Oneskilled in the art would recognize that ligands of other membranereceptors could also be detected in a similar manner. On skilled in theart would recognize that additional membrane proteins could be analyzedin a similar manner, either individually or in multiplexed format.

Example 140 Purification of a Protein

Lipoparticles can be used as a vehicle for the in vitro production ofthe soluble protein sulfated glycoprotein-1 (SGP-1, also known asprosaposin). Conventional protein expression systems often possesslimitations in their ability to produce sufficient quantities of someproteins (in the case of eukaryotic expression systems) or appropriatelyprocessed forms (in the case of prokaryotic expression systems) forstructural analyses such as crystallography. Lipoparticles allow theproduction of large (milligram) quantities of highly concentratedproteins using mammalian cells to ensure physiologically appropriatepost-translational modifications. A Gag/SGP-1 fusion plasmid will beconstructed as previously described. The fusion gene will contain aconsensus enterokinase protease cleavage site (see, for example, Jenny RJ et al. Protein Expr Purif. 2003 September; 31(1):1-11; and Yuan L D etal. Protein Expr Purif. 2002 July; 25(2):300-4.) interposed between theGag and SGP-1 domains. Lipoparticles are produced in HEK-293 cells usingthe Gag/SGP-1 fusion in a manner similar to that described previouslyfor other Gag-fusion proteins. Harvested lipoparticles are lysed in a 1%solution of CHAPSO detergent, and the lysate passed through a sepharoseaffinity column covalently coupled with anti-Gag IgG. After washing withtwo column-volumes of buffer, Gag/SGP is eluted using high salt and/orlow pH elution buffer, and concentrated by a centrifugation filter(Centricon™). The presence and purity of the Gag/SGP in the resultantfiltrate is verified by polyacrylamide gel electrophoresis, and westernblot using anti-Gag and anti-SGP antibodies. SGP can be released fromthe Gag partner as desired by treatment with enterokinase (Roche). Oneskilled in the art would recognize that any soluble protein, such as akinase, phosphatase, or fluorescent protein, could be similarlyexpressed and purified using this technique.

Example 141 Lipoparticle Ligand Fishing

Lipoparticles bound to a bead substrate can be used to capture ligandsfor molecular identification. The identification of potential receptorligands in complex biological mixtures such as blood serum presents amajor challenge in pairing of orphan receptors and ligands. Affinitychromatography is a useful method for such “ligand fishing”applications. The characteristics of Lipoparticles significantlysimplify affinity chromatography techniques for membrane proteins.Lipoparticles containing no specific membrane proteins(‘null’-lipoparticles), and Lipoparticles containing the membranereceptor CCR5 are produced by techniques previously described. Both setsof Lipoparticles are surface-biotinylated as outlined previously.Neutravidin beads (Pierce) are suspended in HBS, biotinylatedLipoparticles are added, and lipoparticles are allowed to bind for 1hour at room temperature. The lipoparticle-coated beads are packed intotwo separate glass columns, one containing the null-Lipoparticles andone containing the CCR5-Lipoparticles, and allowed to equilibrate byflowing through five column-volumes of HBS. Supernatant from the FC36.12cell line (used originally to isolate the chemokines RANTES, MIP1α, andMIP1β as inhibitory factors of HIV (Cocchi, et al. (1995), Science,270:1811-1815)) are applied to the null-Lipoparticle column, and theflow-through is collected. This column removes compounds that bindnon-specifically to the beads and/or non-target components of theLipoparticles. The null-Lipoparticle column can be regenerated bywashing with HBS containing 2M NaCl, or other high salt solutions, or bywashing with pH extremes. The collected flow through is applied to theCCR5-lipoparticle column, washed with five column volumes of buffer, andthe flow through discarded. Molecules that have specifically bound tothe Lipoparticle-incorporated CCR5 are eluted from the column usingbuffer containing 1M NaCl, and collected in fractions. This eluate isanalyzed by SDS-PAGE gel with silver staining, as well as by massspectroscopy, to analyze the protein content of the eluate. Ligandsspecific for CCR5 are isolated. This technique could be used to identifyligands, in a variety of sample formats, for any membrane protein bylinking the appropriate Lipoparticles to the bead substrate. Thetechnique could also be applied to chemical libraries for theidentification of potential pharmaceutical agents that interact withmembrane proteins.

Example 142 Measurement of an Arrayed Library with Lipoparticles

Lipoparticles are coated onto a poly-lysine slide by spraying onto theslide surface a solution of CXCR4-Lipoparticles and 1% sucrose (astabilizer). The Lipoparticles are allowed to dry and are stored at 4 C.until ready to use. When ready for use, the slide is arrayed with alibrary of antibodies using a microarrayer. The antibodies are allowedto bind to the Lipoparticles and are then washed away. The binding ofthe antibodies is then detected by coating the entire slide with afluorescent secondary antibody that recognizes the first antibody. Theslide is washed and spots that have bound antibody are detected byvisualizing fluorescent spots.

One skilled in the art would recognize that the library may be composedof antibodies, hybridoma supernatants, drug candidates, or peptides. Oneskilled in the art would also recognize that the library arrayed ontothe Lipoparticles may also contain glycerol to prevent drying of thespots. Alternatively, Lipoparticles are coated by covering the entireslide with a solution of Lipoparticles in 1% sucrose, allowingLipoparticles to attach for 1 hour, and then removing the Lipoparticlesolution. The Lipoparticles may or may not be allowed to dry.

Example 143 Identifying a Binding Partner Using Phage Display

Immobilized Lipoparticles containing Kv1.3 (Aim 1.3) are used asspecific target molecules to select phage displaying reactive scFVfragments (“panning”). Because phage panning is essentially a ligandbinding reaction, biochemical parameters influencing binding specificityand affinity (e.g. NaCl concentration, pH, etc.) can be manipulated tocontrol the stringency of selection. The values for these parameters areselected on the basis of round of phage selection (later rounds ofselection can be higher stringency to isolate more specific or higheraffinity phage) and the library (phage libraries created for specificantigens can contain larger numbers of phage and with greater bindingaffinities).

In the first round of panning, non-specific binding sites on immobilizedLipoparticles are blocked using Blotto (2% skimmed milk in PBS) and10¹²-10¹³ phage are added. Non-biotinylated Lipoparticles without Kv1.3are also be included during phage binding to competitively adsorb phagethat bind non-specifically to components of the Lipoparticle surface.After allowing binding to proceed for 2 h at room temperature, the beadsare magnetically separated and washed extensively with PBS. Bound phageare eluted by Trypsin digestion, which cleaves the specific scFV-Kv1.3interactions but leaves most non-specific interactions intact (henceimproving phage selectivity), and does not affect phage coat proteinsnecessary for subsequent bacterial infection. The eluted phage arerecovered in their host strain bacteria (TG1 E. coli) and expanded withthe use of a helper strain of phage (KM13 provides phage proteins intrans that are replaced in phage libraries by antibody inserts). Twomore rounds of panning can conducted to select for the most specific andhighest affinity binders.

Phage isolated after the third round of panning are tested forreactivity to cells expressing Kv1.3 on their surface. Cells are grownto confluence in 96-well microplates and transiently transfected with aplasmid expressing Kv1.3. Non-specific binding sites are blocked with 3%BSA in PBS, and isolated phage are added. After removing unbound phageby washing, bound phage are detected using an anti-phage (M13) secondaryMAb coupled to a reporter (HRP). Cells used for screening (quail QT6cells) are of heterologous origin than the cells used to produceLipoparticles (human HEK-293 cells) to reduce the probability ofdetecting phage against unwanted proteins. Cells lacking Kv1.3 are usedas negative controls. Flow cytometry may be used as an alternativedetection method. Phage that react with cells expressing Kv1.3 aresequenced to determine the encoded antibodies, and scFv fragments areisolated from bacterial periplasmic space (expressed as a scFv-pIIIfusion protein from TG-1 E. coli, or as isolated scFv from the HB2151non-suppressor strain of E. coli).

Western blotting is used to determine if the isolated MAbs are directedto linear or conformational epitopes of receptors (MAbs that react withdenatured protein are nearly always to linear epitopes). Lysates of QT6cells expressing Kv1.3 are separated by SDS-PAGE under denaturingconditions. The proteins are transferred to PVDF membranes, which is cutinto strips, and exposed to isolated scFv. Reactivity of a MAb to areceptor by Western blot is indicative of recognition of a linearepitope within the receptor.

One of the most useful functions of any MAb is the ability to recognizeand block important structural and/or functional determinants in atarget protein. This ability to inhibit function directly enableshumanized MAbs to serve as therapeutic agents, a role that polyclonalantibodies, intracellular-epitope antibodies, and linear-epitopeantibodies are typically not capable of performing. The ability ofselected phage to recognize conformational epitopes in Kv1.3-expressingcells is determined from their ability to competitively inhibit ligandbinding. Briefly, quail QT6 cells expressing Kv1.3 are pre-incubatedwith phage-derived scFv's prior to addition of radiolabeledCharybdotoxin. The ability of each phage to competitively inhibitradioligand binding under a variety of conditions is determined. Oneskilled in the art would recognize that phage panning could also beconducted against whole virus or virus-like particles.

Example 144 Detection of GPCR Activation

3 ug CXCR4-Lipoparticles are suspended in a solution of 500 nMFL-GTP-γS. 10 uM melittin peptide is added to porate the Lipoparticles.One skilled in the art would recognize that alternative permeabilizationconditions may also be employed, and that alternative methods forincorporation of FL-GTP-γS can also be used. These include, but are notlimited to, poration using mild sonication or electroporation. Controlscan include Lipoparticles without CXCR4 and CXCR4-Lipoparticles withoutmelittin. To test GPCR signaling, CXCR4-Lipoparticles in a buffercontaining 50 mM Hepes 8.0, 1 mM EDTA, 20 mM MgCl₂, 100 mM NaCl, and 1mM DTT are stimulated with the CXCR4 agonist SDF-1. Stimulation of CXCR4causes Gα to irreversibly bind FL-GTP-γS, allowing the BODIPYfluorophore to emit a detectable fluorescent signal. Fluorescenceemission is not altered in null-Lipoparticles, or in Lipoparticlestreated with a CXCR4 antagonist such as the antibody 12G5. Fluorescenceis measured in real-time, beginning prior to addition of SDF-1, using aPerkin Elmer LS-50B fluorometer (excitation 485, emission 530). Oneskilled in the art would recognize that alternative guanine-nucleotidescould be incorporated into Lipoparticles in a similar manner, includingMANT-GTP, MANT-GMPPNP, BODIPY-FL-GTP, BODIPY-R6G-GTP, BODIPY-TR-GTP,BODIPY FL GMPPNP, BODIPY FL GTP-γ-S thioester, TNP-GTP (2′-(or3′-)O-(trinitrophenyl)guanosine 5′-triphosphate), BzBzGTP (2′-(or3′-)O-(4-benzoylbenzoyl)guanosine 5′-triphosphate), S-(DMNPE-caged)GTP-γ-S, or Europium-GTPγS. One skilled in the art would also recognizethat the Lipoparticles within this example could be modified prior tostimulation with agonist to permit signaling components to interact.Such modification could include, but is not limited to, disruption bysonication, vortexing, or detergent; or poration using melittin,Streptolysin-O, polyethylene glycol, high amounts of calcium, or lowamounts of alkanes. As an alternative to fluorescence emission,fluorescence polarization or FRET could also be used to detect bindingof fluorescent GTPγS to the G protein within the Lipoparticle.

Example 145 Detection of GPCR Activation by FRET

YFP is fused to the carboxy-terminus of CXCR4 and CFP fused to thecarboxy terminus of Gag. Fusion proteins are created using standardcloning methodology. Lipoparticles containing both proteins together areconstructed. The fusion proteins are incorporated into theseLipoparticles as described herein. One skilled in the art wouldrecognize that additional or alternative GPCRs, G proteins, andfluorescent protein labels could similarly be incorporated. One skilledin the art would recognize that the GPCR could contain the CFP proteinand the Gag could contain the YFP protein. In some embodiments, theLipoparticle is also constructed to contain a G protein, for exampleGag-Gprotein. To test their function, the labeled Lipoparticles areexposed to the CXCR4 agonist SDF-1. Ligand binding by CXCR4 causes aconformational change in CXCR4 that induces a change in the relativeproximity of the CFP and YFP tags, causing a change in the fluorescentemission from the CFP-YFP FRET pair. Fluorescence emission is notaltered in null-Lipoparticles, or in Lipoparticles treated with a CXCR4antagonist such as the antibody 12G5. Fluorescence is measured inreal-time, beginning prior to the addition of SDF-1, using a PerkinElmer LS-50B fluorometer.

Example 146 Generation of an Antibody Against a Gag-Fusion Protein

Luciferase is incorporated into Lipoparticles as described herein byfusing luciferase to Gag. Gag-luciferase lipoparticles are produced andpurified. 300 ug of luciferase-containing lipoparticles are injectedinto mice as described herein. Polyclonal and monoclonal antibodies toluciferase-containing lipoparticles are generated as described inexamples herein. The serum is screened for reactivity against luciferaseusing purified luciferase protein, and either polyclonal antibodies willbe collected or hybridomas established for monoclonal antibodyproduction.

Example 147 Creation of an Array of Viruses

HIV-1 virions from the strains JRFL, IIIB, and 89.6 are prepared bypurifying viruses through sucrose cushions. The viruses are resuspendedin 50 mM Hepes 7.0 containing 5% sucrose. Spots of viruses are arrayedon a polylysine slide using a microarrayer. The viruses are allowed todry and the slide is stored at −20 C. until further use. When ready foruse, the slide is brought to room temperature. An antibody against HIV-1Envelope (b12) is used to probe the slide, using a fluorescent secondaryantibody for detection. Reactivity of the antibody with viruses on theslide, a indicated by fluorescent spots, is indicative of the epitopecontained within that strain of HIV-1. Reactivity of a broadlyneutralizing antibody such as b 12 against a panel of diverse HIV-1strains is indicative of the antibody's potency as part of a humoralimmune response against the virus, a critical part of determining a testvaccine's potential. One skilled in the art would recognize that theviruses herein may be modified, such as with a fluorophore, biotin,avidin, streptavidin, or WGA to facilitate attachment and/or detection.One skilled in the art would also recognize that the probe used todetect the virus array may be a monoclonal antibody, a polyclonal serum,a phage, a lipoparticle, a protein, a peptide, an organic molecule, oran inorganic molecule. One skilled in the art would also recognize thatother stabilizers could also be used, including glucose, glycerol,gelatin, or trehalose.

Example 148 Detection of an Array of Binding Partners Using aLipoparticle

An array of antibodies is prepared by microarraying different monoclonalantibodies onto a surface. The array is then probed using afluorescently labeled (Gag-GFP) lipoparticle containing the GPCR CXCR4.Spots that bind the lipoparticle, as visualized by fluorescence of thespot, indicate that CXCR4 has interacted specifically with a MAb at thatspot. The identity of the MAb is known from the location on the array.

One skilled in the art would recognize that the array could consist ofany number of molecules, including monoclonal antibodies, hybridomasupernatant, polyclonal antibodies, serum, phage, other lipoparticles,proteins, peptides, organic molecules, or inorganic molecules. Oneskilled in the art would also recognize that fluorescently labeledviruses or virus-like particles could also be used. Other means oflabeling and detecting the particles could also be used, includingfluorescence, luminescence, radioactivity, or magnetism.

Example 149 Creation of a Microarray of Lipoparticles

Lipoparticles were constructed with either a fluorescent core (Gag-GFP),a fluorescent membrane protein (CXCR4-GFP), or non-fluorescent versionsof the same (Gag and CXCR4). Lipoparticles were prepared and purified asdescribed herein. Sucrose was added to Lipoparticles to a finalconcentration of 5%. Spots of Lipoparticles were then arrayed on apolylysine slide using a microarrayer. The Lipoparticles were allowed todry and were stored at 4 C. until further use. Fluorescence in the greenchannel was visualized by imaging the Lipoparticles with a FITC filteron an AlphaInnotech AlphaArray 7500i (FIG. 30). The slide was thenprobed with a conformation-dependent anti-CXCR4 MAb (447.08). Binding ofthis primary antibody was detected using a Cy3-labeled secondaryantibody. Slides were washed with PBS after incubation with eachantibody. After staining, the array was again visualized using bothgreen and red filters. Green spots indicated that the Lipoparticles werestill bound to the slide, and red spots indicated that CXCR4 within theLipoparticles was present and structurally intact. Control Gag particleswithout CXCR4 or GFP demonstrated little or no background.

The disclosures of each and every patent, patent application,publication, and accession number cited herein are hereby incorporatedherein by reference in their entirety. The appended sequence listing ishereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A lipoparticle comprising an external lipidbilayer; an enveloped retroviral structural protein; and a multiplemembrane spanning protein, wherein said enveloped retroviral structuralprotein is an uncleaved gag protein and wherein the multiple membranespanning protein that is incorporated into the lipoparticle does notbind to the gag protein, provided that the only viral proteins in thelipoparticle are structural proteins.
 2. The lipoparticle of claim 1wherein said multiple membrane spanning protein is a G protein coupledreceptor.
 3. The lipoparticle of claim 1, wherein said Gag is frommurine leukemia virus, rous sarcoma virus, HIV, SIV, avian leukemiavirus, or equine anemia virus.
 4. A composition comprising an isolatedlipoparticle of claim 1 attached to a biosensor surface.
 5. Thelipoparticle of claim 1, further comprising a G-protein.
 6. Thelipoparticle of claim 5, wherein said G-protein is a modified G-protein.7. The lipoparticle of claim 6, wherein said modified G-proteincomprises a fusion protein.
 8. The lipoparticle of claim 7, wherein saidfusion protein comprises a fluorescent protein, a linker, a viralprotein, membrane protein, a protease cleavage sequence, or combinationsthereof.
 9. The lipoparticle of claim 2, wherein said G protein coupledreceptor (GPCR) is a modified GPCR.
 10. The lipoparticle of claim 9,wherein said modified GPCR comprises a fusion protein.
 11. Thelipoparticle of claim 10, wherein said fusion protein comprises afluorescent protein, a linker, a viral protein, membrane protein, aprotease cleavage sequence, or combinations thereof.
 12. Thelipoparticle of claim 5 further comprising a GTP analog.
 13. Thelipoparticle of claim 12 wherein said GTP analog is a fluorescent GTPanalog.
 14. An immunogen comprising a lipoparticle according to claim 1.15. A method of eliciting an immune response to a multiple membranespanning protein, said method comprising the introduction of thelipoparticle of claim 1 to an animal.
 16. The lipoparticle of claim 1,wherein said multiple membrane spanning protein is an ion channelprotein.
 17. The lipoparticle of claim 1, wherein said multiple membranespanning protein is a transporter protein.
 18. A lipoparticle comprisingan external lipid bilayer; an enveloped retroviral structural protein;and a multiple membrane spanning protein, wherein said envelopedretroviral structural protein is an unmodified, uncleaved gag proteinand, provided that the only viral proteins in the lipoparticle arestructural proteins.
 19. A lipoparticle comprising an external lipidbilayer; an enveloped retroviral structural protein; and a multiplemembrane spanning protein, wherein said enveloped retroviral structuralprotein is an uncleaved gag protein and wherein said gag protein doesnot comprise a heterologous tag, provided that the only viral proteinsin the lipoparticle are structural proteins.